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Multitarget anti-Alzheimer compounds 1-3, designed by combining a naphthoquinone function and a tacrine fragment, displayed excellent in vitro AChE (AcetylCholinEsterase) inhibitory potencies and proved to be effective as Abeta (Amyloidbeta) anti-aggregants. The x ray analysis of 2 in complex with AChE allowed rationalizing the outstanding activity data, IC50 = 0.72nM. The non toxic derivatives 2 and 3: (i) completely reverted the decrease in viability induced by Aß in immortalized cortical neurons; (ii) showed antioxidant activity in human glioma; and (iii) crossed the blood-brain barrier.

As a part of an investigation on molecular hybrids as new serine protease inhibitors, the pyrazolo [4,3-c][1,2,5]oxadiazin-3(5H)-one ring system was selected as a model of potential mechanism-based inhibitors. Due to the inherent reactivity of this system an optimal balance between susceptibility to nucleophilic attack and stability in solvents was sought prior to development as therapeutic agents. Substitutions on N5 and C7 of the supporting pyrazole ring with either aliphatic or aromatic groups (compounds 2 a-m) and the replacement of the carbonyl oxygen on the reactive oxadiazinone ring with sulfur (compounds 3a,i) were explored. Two members (2i and 2k) of this class of inhibitors displayed time-dependent inhibition of HLE suggesting mechanism-based inhibition. The observation that HLE generated a product(s) from compound 2i which displayed an identical UV-Visible spectrum to that observed during non-enzymatic hydrolysis further supports this proposal. FlexX-based docking of these compounds into a model of the human leukocyte elastase (HLE) active site produced a molecular model of the inhibitor-enzyme interaction.

Synopsis Oxidation hair dyes as raw materials may present toxic impurities from synthesis or degradation pathways, which are responsible for allergic phenomena. In the present study selective and sensitive HPLC methods are proposed for the determination of Bandrowski base, 4-chloroaniline, 1,2-phenylenediamine and 4,4'-diaminoazobenzene in 1,4-phenylenediamine as well as 2,4-diaminoanisole in N-hydroxyethyl-4-methoxy-1,3-phenylenediamine. The proposed methods, based on reversed phase mode separation using 1,8-diaminooctane as amine modifier and photodiode array detection, proved to be suitable for the purity assessment of these widely used hair dyes; in particular the cited potential impurities were determined at ppm level.

A series of 12 chiral arylcarboxylic acids were chromatographed on an immobilized human serum albumin chiral stationary phase (HSA-CSP). The effects of solute structure on chromatographic retentions and enantioselective separations were examined by linear regression analysis and the construction of quantitative structure-enantioselective retention relationships. Competitive displacement studies were also conducted using R-ibuprofen as the displacing agent. The results indicate that the enantioselective retention of the solutes takes place at the indole-benzodiazepine site (site II) on the HSA molecule and that chiral recognition is affected by the hydrophobicity and steric volume of the solutes. The displacement studies also identified a cooperative allosteric interaction induced by the binding of R-ibuprofen to site II.

Derivative and difference spectrophotometric methods are described for the direct simultaneous analysis of combinations of Trimethoprim with sulfonamide drugs (sulfadiazine, sulfamethoxazole, sulfamethoxypyridazine) in commercial dosage forms. The more advantageous approach (derivative and difference mode) is suggested for each binary mixture. The assay results are compared with those obtained by a new chromatographic (HPLC) method, involving a C-18 column and a mobile phase (pH 6.5) containing 1.8 diaminooctane as amine modifier and sodium heptansulfonate as ion pairing agent.

The synthesis, diastereomeric separation, assignment to erythro- and threo-configuration by 1HNMR, and optical resolution of 3-(p-trifluoromethyl-phenyl)- and (p-thioanisyl)-2-biphenylyl-3-hydroxypropionic acids are described. The enantiomers were submitted to a preliminary assay to determine antiinflammatory activity.

The optically active diastereometric erythro- and threo-3-thienyl-analogues 6a-d of 3-aryl-2-biphenylyl-3-hydroxy-propionic acids have been prepared. Some stereoisomers of 3-(3-thienyl)derivative 6b showed inhibition around 40% in the carrageenan-induced rat paw edema test.

In another paper1 we have reported the synthesis of cligomeric poly(oxyethyleneglyccl) derivatives of the title compound. One of these derivatives was orally administered to human volunteers, and proved to be able to give much higher and more sustained blood levels of Urscdecxycholic acid (UDCA) than the free acid, if given in equivalent doses. This prompted us to study also high molecular weight polymeric derivatives of UDCA.

The synthesis and pharmacological evaluation of Oligomeric and polymeric derivatives of drugs is receiving increasing attention by several groups 1-9. Usually, in these derivatives, the drug moieties are linked to a proper oligomeric or polymeric matrix by means of covalent bonds of limited stability to biological environments. This may ensure a gradual release of the active principle after introduction into the body Fluids. Poly(oxyethyleneglycol)s, as matrices, appear to be particularly promising on this respect7.

The treatment of Alzheimer's disease (AD) still remains an area of significant unmet need, with drugs that only target the symptoms of the disease. Therefore, there is considerable need for disease-modifying therapies. The complex etiology of AD prompts scientists to develop multitarget strategies to combat causes and symptoms. To this aim, we designed, synthesized, and tested four new carbamates as dual cholinesterase-FAAH inhibitors. The dual activity of these compounds could lead to a potentially more effective treatment for the counteraction of AD progression, because they would allow regulation of both ACh and eCB signaling and improve neuronal transmission and/or counteract neuroinflammation.

Lipoic acid (LA) is a natural antioxidant. Its structure was previously combined with that of the acetylcholinesterase inhibitor tacrine to give lipocrine (1), a lead compound multitargeted against Alzheimer's disease (AD). Herein, we further explore LA as a privileged structure for developing multimodal compounds to investigate AD. First, we studied the effect of LA chirality by evaluating the cholinesterase profile of 1's enantiomers. Then, a new series of LA hybrids was designed and synthesized by combining racemic LA with motifs of other known anticholinesterase agents (rivastigmine and memoquin). This afforded 4, which represents a step forward in the search for balanced anticholinesterase and antioxidant capacities.

The multifactorial nature of Alzheimer’s disease (AD) offers us a textbook example where parental compounds, mostly marketed, are modified with the aim of improving and/or conferring two or even more biological activities to contrast or less frequently revert the disease’s symptoms. This is the case of tacrine and its dimeric derivative bis (7)-tacrine which, for instance, paved the way for the development of a broad collection of very interesting homo- and heterodimeric structures, conceived in light of the emerging multi-target approach for AD-related drug discovery. As a contribution to the topic, we report here the design, synthesis and biological evaluation of 12 compounds referable to bis (7)-tacrine. In addition to the cholinesterase activity, some of the selected compounds ( 7 – 9 and 12 ) were capable of inhibiting the non-enzymatic function of AChE and/or showed a remarkable activity against BACE1. Thus, the present study outlines a series of newly synthesized molecules, structurally related to bis (7)-tacrine, endowed with extended biological profile in agreement with the emerging multi-target paradigm.

Beta-Secretase 1 (BACE1) is the enzyme involved in the abnormal production of the amyloidogenic peptide Abeta42, one of the major causes of histological hallmarks of Alzheimer's disease. Thus, BACE1 represents a key target protein in the development of new potential drugs for the non-symptomatic treatment of Alzheimer's disease. Since the discovery of BACE1 one decade ago, both in the pharmaceutical industry and in academia there has been an intense search for novel inhibitors to be developed as new effective drugs. There is a great deal of interest in the discovery of selective non-peptide BACE1 inhibitors with a new chemical skeleton, suited for central nervous system penetration and endowed with more appropriate pharmacokinetic properties. Therefore, the selection of appropriate methods for screening and characterization of BACE1 inhibitors is crucial. This review focuses on the description of the in vitro methods to test BACE1 activity and inhibition, with particular emphasis on fluorescence resonance energy transfer (FRET) methods, aiming at critically highlighting advantages and drawbacks. An overview of BACE1 inhibitors is given, underlying the variability of the FRET methods reported in the literature, and the structure evolution of inhibitors active in cellular cultures and in vivo, from peptide to small synthetic and natural structures.

New stable [6,6]-methano cycloadducts and fulleropyrazolines containing electron-withdrawing groups have been obtained by the reaction of ethyl diazopiruvate with [60]fullerene. The results obtained by a sistematic study conducted both in thermal and catalytic conditions have provided crucial indications concerning the mechanism of this important cluster opening process in fullerene chemistry.

A cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method for the determination of the most important potential impurities of methotrexate (MTX): 2,4-diamino-6-(hydroxymethyl)pteridine, aminopterine hydrate, 4-[N-(2-amino-4-hydroxy-6-pteridinylmethyl)-N-methylamino] benzoic acid, 4-[N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino] benzoic acid, and the distomer D-MTX is presented. The MEKC separation of these compounds was optimized by applying a step-by-step approach. The addition of beta-CD to a conventional MEKC system, based on sodium dodecyl sulfate (SDS) as surfactant, showed to be essential for the enantioresolution of racemic MTX as well as for the separation of the achiral impurities. To achieve high-resolution factor between the peaks adjacent to the main component (L-MTX), as required in the analysis of related impurities, the separation conditions were stressed; in particular, the addition of methanol to the CD-MEKC system resulted in a very effective choice. Under the optimized final conditions (100 mM SDS and 45 mM beta-CD in a mixture of 50 mM borate buffer, pH 9.30-methanol (75:25 v/v)), the method was validated showing a general adequate accuracy (93-106% recovery) in the determination of L-MTX related substances at the impurity level of 0.12% w/w with a relative standard deviation (RSD)% lower than 8% (n = 4). The method was successfully applied to the analysis of pharmaceuticals (tablets and injections) which showed to contain the distomer D-MTX as major impurity and aminopterine hydrate as a further related substance in the commercial tablets.

Amyloid beta 25–35 [Aβ (25–35)], as a peptide model for full-length Aβ in structural and functional investigations, has been chosen for aggregation studies. The complexity of the Aβ (25–35) aggregation process required a multi-methodological analytical approach to obtain reliable and reproducible results. Here, we describe the results obtained by the use of mass spectrometry (MS) for the structural characterization of the self-assembly species during the aggregation process and for the definition of the self-assembly kinetics and myricetin inhibition patterns, comparing the results with those obtained by using the well-established spectroscopic method based on thioflavin T fluorescence. Flow injection electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS) was applied to monitor the disappearance of the monomer specie in the first steps, whereas matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-ToF-MS) was used to follow monomer and small oligomer self-assembly trends in the early stages of the nucleating process.

Objectives: Develop single molecules able to target multiple distinct AD hallmarks. Multi-target directed ligands (MTDLs) should bring the benefit of a synergistic enhancement of therapeutic effects compared to a single-target acting drug. Methods: To achieve MTDL we combined the scaffold of the known acetylcholinesterase (AChE) inhibitor tacrine with a benzofuran unit through a methylene chain. Tacrine-benzofuran hybrids were synthesized from substituted salicylaldehydes and 2-aminobenzoicacid as the available starting materials. The influence of hybrids on the hydrolytic activity of human recombinant AChE and butyrylcholinesterase from human serum were in vitro evaluated by Ellman’s assay while inhibitory potency against human recombinant beta-secretase was studied by a FRET assay using M-2420 as substrate. Inhibition of AChE-induced beta-amyloid(1-40) aggregation and spontaneous beta-amyloid(1-42) aggregation was investigated by a Thioflavin T-based fluorescence assay. Results: Promising new tacrine-benzofuran hybrids, which could be further developed for an effective treatment of AD, were designed, synthesized and in vitro characterized. In particular, the most interesting derivatives within this series showed inhibitory potency against human AChE in the low nanomolar range, a mixed-type mechanism of inhibition, which has been associated with the ability of partially inhibit AChE-induced Aβ fibril formation, were able to significantly inhibit amyloid self-aggregation (at x0.2 concentration) and resulted in potent beta-secretase inhibitors with inhibitory potency in the low micromolar range. Conclusions: The new tacrine-benzofuran hybrids showed an MTDL profile and were able to reduce the hydrolytic activity of human AChE, to potentially prevent amyloid production through the inhibition of human beta-secretase activity and interfere with amyloid aggregation.

Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2×6 mm i.d.), under a three-solvent gradient elution mode and UV detection at 256 nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples.

The plant kingdom is an endless source of chemically diverse bioactive compounds, which are used in traditional folk medicine for the treatment of a wide array of diseases. A previous investigation on the bioactive phytocomponents present in the methanol extract of Phyllanthus muellerianus (PMME) demonstrated an interesting activity against C. sporogenes (MIC= 100 µg/ml) and S. pyogenes (MIC= 300 µg/ml) [1], which supported the traditional use of the extract by local populations in Cameroon. Looking for activity beyond the claimed traditional use [2], PMME was evaluated on human cholinesterase enzymes, namely acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) as selected targets for Alzheimer’s disease (AD) treatment. Indeed AChE inhibitors derived from natural products (galanthamine and rivastigmine) are currently licensed to alleviate cognitive symptoms in dementia. Due to the limited available pharmacological treatments for AD, extensive research has been directed towards the identification of other AChE inhibitors, arising from the plant kingdom. A rational basis for our investigation on the bark extracts of Phyllanthus muellerianus is related to a study carried out in 2007 by Joshi e Parle, in which the antiamnesic potential for Phyllanthus amarus in mice was demonstrated [3]. Anticholinesterase activity was in vitro evaluated by Ellman’s assay [4] using recombinant human AChE and BuChE from human serum. Due to the interesting activity found for the de-fatted PMME (% inhibition at 100 g mL-1 on hAChE = 52.6 ± 0.6, on hBuChE = 70.1 ± 3.1%, n=4), PMME was fractionated by flash chromatography affording six fractions (PMF1-6); PMF1, PMF2 and PMF4 significantly inhibited human cholinesterases, thus they were further purified by a RP-18 solid phase extraction. The bio-guided fractionation allowed the identification of three active phytocomponents, with different selectivities against the two ChEs. Two out of the three bioactives have been isolated so far: compound A from PMF1 and compound B from PMF4, both alkaloids. Compound A showed to be a hBuChE selective inhibitor (IC50 = 44.8 ± 3.0 g mL-1 and IC50 = 382 ± 15 g mL-1, for hBuChE and hAChE, respectively) while compound B showed comparable inhibitory potencies against both enzymes (IC50 = 5.45 ± 0.20 g mL-1 and IC50 = 12.6 ± 0.4 g mL-1, for hAChE and BuChE, respectively). Compared to the commercially available AD drug galanthamine, compound B is one order of magnitude less active on hAChE (galanthamine IC50 = 0.6 g mL-1), representing, however, a potential new natural scaffold to undergo towards optimization. The chemical structure of the two isolated alkaloids is under investigation by 13C and 1H-NMR. Moreover, the isolation of compound C in suitable amount for the evaluation of the biological profile and its structural elucidation has being carried out. References [1] G. Brusotti, I. Cesari, G. Frassa, P. Grisoli, C. Dacarro, G. Caccialanza, J. Ethnopharmacol., 2011, 35, 797–800. [2] R. Brisson, Etudes pygmées, SELAF n 376, Ed Peeters, 1999, Paris. [3] H. Joshi and M. Parle, African Journal of Biomedical. Research., 2007, 10, 165–173. [4] G.L. Ellman, K.D. Courtney, V. Jr. Andres, R.M. Feather-Stone, Biochem. Pharmacol., 1961, 7, 88-95

L'analisi volumetrica o volumetria comprende vari metodi di analisi quantitativa, tutti caratterizzati dall'applicazione di un procedimento pratico definito con il termine "titolazione". Si tratta di un procedimento che permette di determinare la quantità di un analita in una soluzione (campione o titolato), basandosi sulla misura del volume di un reagente a concentrazione nota (titolante) che viene aggiunto al campione fino a che la sua reazione con l'analita arriva a completamento.

Progression of Alzheimer’s disease involves aggregation of amyloid-beta (Abeta) peptides, a complex process that involves the formation of several soluble intermediates and ends up with the deposition of ordered fibrillar architectures. The determination of the Abeta42 selfassembly species targeted by an inhibitor is a key step in the identification of new inhibitors endowed with a suitable profile. In this context, the subtle characterization of myricetin (Myr)mode of action at a molecular level was performed by using different MS techniques, which allowed the monitoring of the modifications induced by Myr on the first occurring Abeta42 self-assembly species. Results showed a persistent level of monomer and decreased formation of ordered Abeta42 aggregates in the presence of Myr, further, nano-LC-nano-ESIQTOF, MALDI-TOF, and ESI-IT highlighted the formation of a new oxidizedAbeta42 species, which is less prone to aggregation, in the presence of Myr. Coupling tryptic digestion and nano-LC-nano-ESI-QTOF also allowed the identification of Met35 as the specific site of oxidation along the Abeta aminoacid chain. Therefore, the detailed investigation by different MS techniques allowed better understanding of the molecular modification at the basis of the antiaggregating properties of Myr and highlighted its oxidizing action on the residue Met35 in Abeta monomers.

Human acetylcholinesterase (AChE) is a widely studied target enzyme in the development of therapeutic agents for the treatment of Alzheimer’s disease (AD). AChE inhibitors have been for a long time the only drugs available on the market for the symptomatic treatment of this widespread pathology and still represent 75% (3 out of 4) of marketed drugs. The interest on AChE inhibitors has evolved in last two decades after AChE’s peripheral binding site (PAS) was hypothesised to promote the deposition of the neurotoxic -amyloid peptide (A).1 Therefore, in the light of this non cholinergic activity, new AChE inhibitors, able to prevent the interaction between AChE’s PAS and A, have been designed and investigated. Miniaturization and automation of the screening system will greatly implement the drug discovery process for AD, in which a large number of new chemical entities needs to be screened for affinity towards a specific target, such as AChE. On the light of these premises, in this talk, two different approaches to the development of screening tools for the investigation of compounds able to bind to the CAS (catalytic anionic site) and PAS of human AChE will be presented. In particular, first, the development and application of AChE-based enzyme reactors for the automated and rapid screening of inhibitors at the CAS will be described. These bioreactors have been obtained by the covalent immobilization of the target enzyme on a suitable monolithic chromatographic material, inserted into a HPLC system and used for the evaluation of the inhibitory activity and mechanism of action of new ChE inhibitors.2, 3 Then, the initial development of a new AChE-based fluorescence sensing surface for the identification of PAS binders through propidium displacement studies will be presented. The initial selection of the suitable multilayered material, and of the optimal pattern thickness required to maximize fluorescence signal and maintain chemical stability will be also discussed. Then, the selective immobilization of recombinant human AChE on the SiO2 architectures with optimal geometry and chemistry will be shown. Thanks to the combined use of atomic force microscopy and CLSM it was demonstrated that the enzyme distribution selectively matched with the initial SiO2 features (independently from their shapes and dimensions). In the optimal design, the AChE-based biosensing surface showed an efficient fluorescence emission after labelling with propidium, a selective fluorescent probe of the peripheral binding site of the AChE.4 1. Inestrosa, N. C.; Alvarez, A.; Perez, C. A.; Moreno, R. D.; Vicente, M.; Linker, C.; Casanueva, O. I.; Soto, C.; Garrido, J. Neuron 1996, 16, 881-91. 2. Bartolini, M.; Cavrini, V.; Andrisano, V. J Chromatogr A 2007, 1144, 102-10. 3. Bartolini, M.; Greig, N. H.; Yu, Q. S.; Andrisano, V. J Chromatogr A 2009, 1216, 2730-8. 4. Bartolini, M.; Naldi, M.; Nicolau, D. V.; van Delft, F. C.; van Zijl, J.; Snijder, J.; van den Heuvel, E. F.; Naburgh, E. P.; Calonghi, N.; Andrisano, V. Anal Bioanal Chem 2012.

Simulating protein flexibility is a major issue in the docking-based drug-design process for which a single methodological solution does not exist. In our search of new anti-Alzheimer ligands, we were faced with the challenge of including receptor plasticity in a virtual screening campaign aimed at finding new β-secretase inhibitors. To this aim, we incorporated protein flexibility in our simulations by using an ensemble of static X-ray enzyme structures to screen the National Cancer Institute database. A unified description of the protein motion was also generated by computing and combining a set of grid maps using an energy weighting scheme. Such a description was used in an energy-weighted virtual screening experiment on the same molecular database. Assessment of the enrichment factors from these two virtual screening approaches demonstrated comparable predictive powers, with the energy-weighted method being faster than the ensemble method. The in vitro evaluation demonstrated that out of the 32 tested ligands, 17 featured the predicted enzyme inhibiting property. Such an impressive success rate (53.1%) demonstrates the enhanced power of the two methodologies and suggests that energy-weighted virtual screening is a more than valid alternative to ensemble virtual screening given its reduced computational demands and comparable performance. © 2012 American Chemical Society.

In this work, we report a rational structure-based approach aimed at the discovery of new 2-aminoimidazoles as β-secretase inhibitors. Taking advantage of a microwave-assisted synthetic protocol, a small library of derivatives was obtained and biologically evaluated. Two compounds showed promising activities in both enzymatic and cellular assays. Moreover, one of them exhibited the capability to cross the blood-brain barrier as assessed by the parallel artificial membrane permeability assay. © 2011 Elsevier Ltd. All rights reserved.

The complex etiology of Alzheimer's disease prompts scientists to develop multi-target strategies to combat causes and symptoms. In line with this modern paradigm and as a follow-up to our previous studies, we designed and synthesized a focused collection of new 2-arylbenzofurans and evaluated their biological properties towards specific targets involved in AD, namely human AChE and human BuChE, and Aβ fibril formation. Selected compounds were also tested for their ability to inhibit Aβ neurotoxicity in terms of neuronal viability loss, and to prevent Aβ peptide-binding to cell membrane and intracellular reactive oxygen species (ROS) formation. The different modifications introduced in the structure of our lead compound led to an increase in activity towards one or more of the selected targets: the anticholinesterase activity of some compounds was found to be significantly higher than previously obtained related molecules, and the compounds also proved to possess Aβ anti-aggregating properties and neuroprotective effects. The most interesting multi-target compounds were 18, and 1. Interestingly, 1 also showed good selectivity and moderate affinity for CB1 receptor, opening new perspectives in the field of research on AD, since cannabinoid ligands have been widely reported to have neuroprotective properties.

Extensive evidence suggests that the self-assembly of amyloid-beta peptide (Abeta) is a nucleation-dependent process that involves the formation of several oligomeric intermediates. Despite neuronal toxicity being recently related to Abeta soluble oligomers, results from aggregation studies are often controversial, mainly because of the low reproducibility of several experimental protocols. Here a multimethodological study that included atomic force microscopy (AFM), transmission electron microscopy (TEM), fluorescence microscopy (FLM), mass spectrometry techniques (matrix-assisted laser desorption/ionization time-of-flight [MALDI-TOF] and electrospray ionization quadrupole time-of-flight [ESI-QTOF]), and direct thioflavin T (ThT) fluorescence spectroscopy were enabled to set up a reliable and highly reproducible experimental protocol for the characterization of the morphology and dimension of Abeta 1-42 (Abeta42) aggregates along the self-assembly pathway. This multimethodological approach allowed elucidating the diverse assembly species formed during the Abeta aggregation process and was applied to the detailed investigation of the mechanism of Abeta42 inhibition by myricetin. In particular, a very striking result was the molecular weight determination of the initial oligomeric nuclei by MALDI-TOF, composed of up to 10 monomers, and their morphology by AFM.

The leading mechanistic theory of Alzheimer’s disease (AD) is the “amyloid hypothesis” which states that the accumulation of the amyloid β protein (Aβ), and its subsequent aggregation into plaques, is responsible for the initiation of a cascade of events resulting in neurodegeneration and dementia. The anti-amyloid disease-modifying approach, based on the decrease in the production of Aβ, gained thus a paramount importance. The aim of this study was the design and synthesis of a new series of acetylcholinesterase inhibitors (AChEIs) endowed with anti-Aβ aggregating capability. These dual binding inhibitors, being able to interact both with the peripheral anionic site (PAS) of AChE and the catalytic subsite, proved to be able to inhibit the AChE-induced Aβ aggregation. Thus, starting from the lead compound 1, an AChEI composed by a benzophenone scaffold and a N,N′-methylbenzylamino group, a substantial modification aimed at targeting the PAS was performed. To this aim, different amino-terminal side chains were incorporated into this main framework, in order to mimic the diethylmethylammonium alkyl moiety of the pure PAS ligand propidium. The synthesized compounds proved to effectively and selectively inhibit AChE. Moreover, compounds 16a–c and 18a,b, with a propoxy and a hexyloxy tether respectively, showed a good activity against the AChE-induced Aβ aggregation. In particular, molecular modeling studies confirmed that compounds carrying the diethylaminopropoxy and the diethylaminohexyloxy side chains (compounds 16a and 19a, respectively) could suitably contact the PAS pocket of the enzyme.

The degradation of ascorbic acid (AA) has been considered one of the causes of quality and color changes during processing and storage of cosmetic products. The degradation process of AA is very complex and proceeds toward a number of oxidation/reduction and intermolecular rearrangement reactions [1]. In particular, AA is highly unstable in aqueous solution with the formation of degradation products such as dehydroascorbic acid, 2-furoic acid, 3-hydroxy-2-pyrone, furfural, etc. The nature of the degradation products depends on the reaction conditions. In the presence of oxygen (aerobic) the oxidation of ascorbic acid leads to the formation of several intermediates [2] and the final formation of various five carbons compounds such as 2-furoic acid and 3-hydroxy-2-pyrone. Under anaerobic conditions ascorbic acid was found to degrade via several steps to furfural [3]. The aims of our study was to investigate the AA stability in an aqueous cosmetic formulation, containing resveratrol (R), melatonin (M). The degradation kinetics of AA have been studied in single solutions, in the presence of resveratrol and melatonin, and in the commercial sample at different temperature (25, 40 and 60 °C). A reverse phase chromatographic method with gradient elution was developed by using both UV and mass spectrometric detection (HPLC-UV, LC-ESI-MS). The separation of the components of the cosmetic solution and the main degradation products (2-furoic acid, 3-hydroxy-2-pyrone, furfural, dehydroascorbic acid) was achieved on a Phenomenex LUNA C18 (4,6 x 150 mm ID; 5m) column. It was found that the main degradation products were deriving from the degradation of AA, and that their relative abundance and formation rates were influenced by the presence of M and R. In particular, in the absence of melatonin and resveratrol the degradation led to the preferential formation of dehydroascorbic acid, 2-furoic acid and 3-hydroxy-2-pyrone, representative aerobic degradation products. On the other hand, M and R presence in solution caused a slower AA degradation rate and the preferential formation of furfural, typical anaerobic reaction product. In aqueous solution at 40°C, the contemporary presence of R and M decreased AA degradation rate to t1/2 =267 days, instead of 66 days when AA was alone, demonstrating a protective role in AA degradation . Since R and M are potent antioxidant and free radical scavenger [4,5], it can be hypothesized that they protect AA from oxidation process and promote AA anaerobic degradation to furfural.

Protein misfolding and aggregation has been related to several human disorders, generally termed protein aggregation diseases. These diseases include neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases and peripheral disorders such as systemic amyloidosis and type 2 diabetes. The complexity of the aggregation processes and the intertwined events account for the fact that no effective disease-modifying treatments for these disorders are currently available. Nevertheless, in-depth research into the aggregation processes has recently yielded major insights into some key mechanisms of aggregation-mediated cell toxicity, offering new targets for drug development. In addition, recent findings in the field have identified similar features, revealing the possibility of shared mechanisms and hence potential common approaches for intervention. This review aims to give an overview of potential strategies for tackling protein aggregation and its associated toxicity, focusing on protein aggregation in human disease.

Surfaces that modulate the behaviour of surface-immobilised biomolecules have been of critical interest in the last decades, with applications in biomedical microdevices. Recently, the advances in nanofabrication also allowed the contemplation of a leap-through in the form of the design and fabrication of biomimetic surfaces that replicate the molecular surfaces of large biomolecular structures such as cytoskeleton aggregates. The process of formation of these long-range ordered nanostructures have enormous biological interest, but increasingly they are good examples of ‘fabrication’ processes leading to large nanostructured areas with the design embedded in their smaller components. To this end, we report here an atomic force microscopy (AFM) study of the high order self assembly of F-actin on mica. Actin is one of the principal structural proteins in eukaryotic cells and is an ideal biopolymer for investigations of new modes of higher order self-assembly. G-actin monomer can be polymerized into long right-handed double helical filaments (F-actin) whose formation is induced by Mg2+, K+, Na+, and ATP. F-actin can be considered as a semiflexible and highly charged polyelectrolyte, with diameter DA ~ 8 nm and persistence length of 10 μm [1]. Atomic force microscopy (AFM) is a useful tool for elucidating the topography of biomolecules-covered surfaces, including proteins, and mica is commonly used as a substrate for AFM imaging at molecular resolution due to its atomically-flat surface. In our previous [2] and present work, we visualised different morphologies of assemblies of actin filaments adsorbed on mica and silicon surfaces with different geometries and physical-chemical properties. However, it is notoriously difficult to immobilize negatively charged samples as actin (theoretical pI 5.23) because the electrostatic repulsion between the negatively charged mica surface and the actin assembly results is a weak immobilisation of the filaments, thus rendering the AFM scanning very difficult and prone to artefacts. On silicon surfaces large, flat and long F-actin fibre bundles were visualised, which could indicate a substrate repulsion that favours the assembly of fibres in the bulk and/or the vicinity of the surface, followed by deposition of pre-‘fabricated’ F-actin rafts on the silicon surface. To counter-act the technological problem described above, mica was chemically treated with Mg2+ to increase the affinity towards F-actin. Interestingly, this treatment rendered the mica surface more hydrophobic. On this treated surface we could visualise with great reproducibility F-actin patterns by both contact and tapping mode AFM approaches (Figures 1 and 2). The most interesting aspect was the capability of fabrication ordered patterns formed by F-actin filaments, through the balanced interplay between F-actin self-assembly forces and forces applied by the AFM tip in a contact mode. More specifically, increasing the force applied by the AFM tip we could observe the shift from the visualisation of individual actin filaments (Figure 3, “e” surface) to parallel actin filaments ‘rafts’ (Figure 1 and Figure 3, surfaces “a” and “c”). In ‘visualisation-only’ contact mode, the average height F-actin filaments above the mica surface was 5.5 nm (Figure 2), consistent with the theoretical value. The spacing between rows was found to be a function of the applied tip force, as can be seen in Figure 2 where three different regions were scanned at forces ranging from 7 to 20 nN. Further increasing the force applied by the AFM tip to 50 nN we could clean the mica surface and ‘dragged’ the filaments, still in an ordered manner, i.e., in a direction perpendicular to their axis (Figure 3, transition from surface “d” to surface “b”). Future work will study the parallel characterization of nanostructured solid surfaces which can modulate, either enhancing or retarding, the oligomerisation and fibrillization of actin filaments, w...

We report on a series of hybrid compounds structurally derived from donepezil and AP2238. This study was aimed at improving the activities of the reference compounds, donepezil and AP2238, and at broadening the range of activities of new derivatives as, due to the multifactorial nature of AD, molecules that modulate the activity of a single protein target are unable to significantly modify the progression of the disease. In particular, the indanone core from donepezil was linked to the phenyl-N-methylbenzylamino moiety from AP2238, through a double bond that was kept to evaluate the role of a lower flexibility in the biological activities. Moreover, SAR studies were performed to evaluate the role of different substituents in position 5 or 6 of the indanone ring in the interaction with the PAS, introducing also alkyl chains of different lengths carrying different amines at one end. Derivatives 21 and 22 proved to be the most active within the series and their potencies against AChE were in the same order of magnitude of the reference compounds. Compounds 15, 21-22, with a 5-carbon alkyl chain bearing an amino moiety at one end, better contacting the PAS, remarkably improved the inhibition of AChE-induced Abeta aggregation with respect to the reference compounds. They also showed activity against self-aggregation of Abeta(42) peptide, the most amyloidogenic form of amyloid produced in AD brains, while the reference compounds resulted completely ineffective.

A novel liquid chromatographic method has been developed for use in throughput screening of new inhibitors of human recombinant beta-amyloid precursor protein cleaving enzyme (hrBACE1). The approach is based on the use of an immobilized enzyme reactor (IMER) containing the target enzyme (hrBACE1-IMER) and uses fluorescence detection. The bioreactor was prepared by immobilizing hrBACE1 on an ethylendiamine (EDA) monolithic disk (CIM) and a fluorogenic peptide (M-2420) containing the beta-secretase site of the Swedish mutation of amyloid precursor protein (APP) was used as substrate. After injection into the hrBACE1-IMER system, M-2420 was enzymatically cleaved, giving rise to a fluorescent methoxycoumaryl-fragment (Rt=1.6min), which was separated from the substrate and selectively detected at lambda(exc)=320 and lambda(em)=420nm. Product and substrate were characterized by using a post monolithic C18 stationary phase coupled to an ion trap mass analyser. A calibration curve was constructed to determine the immobilized hrBACE1-IMER rate of catalysis and kinetic constants. Specificity of the enzymatic cleavage was confirmed by injecting the substrate on a blank CIM-EDA. The proposed method was validated by the determination of the inhibitory potency of five reference compounds with activities ranked over four order of magnitude (four peptidic inhibitors and a green tea polyphenol, (-)gallocatechin gallate). The obtained results were found in agreement with the data reported in literature, confirming the validity and the applicability of the hrBACE1-IMER as a tool for the fast screening of unknown inhibitors (more than 6 compounds per hour). Moreover, the hrBACE1-IMER showed high stability during the analysis, permitting its use for more than three months without affecting enzyme activity.

Protein misfolding and aggregation has been related to several human disorders, generally termed protein aggregation diseases. These diseases include neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s diseases and peripheral disorders such as systemic amyloidosis and type 2 diabetes. The complexity of the aggregation process and the intertwined events account for the fact that no effective disease-modifying treatment is currently available for these disorders. Nevertheless, in-depth research into the aggregation processes has recently yielded major insights into some key mechanisms of aggregation-mediated cell toxicity, offering new targets for drug development. A multiple-methodological study including atomic force microscopy (AFM), mass spectrometry techniques (MALDI-TOF, ESI-IT, ESI-Q-TOF), direct Thioflavin T fuorescence spectroscopy, transmission electron microscopy (TEM), fluorescence microscopy (FLM), is here presented, which enables the characterization of the morphology, dimension and molecular weight of aggregates in the self-assembly kinetics. This approach is useful to elucidate in detail the diverse assembly species formed in amyloid peptides folding process. Different orthogonal techniques resulted suitable to obtain technique-independent outcomes. Moreover, MALDI–TOF mass spectrometry allowed the detection and characterization of the very early stages and nucleating species of the self-assembly process. This multi-methodological approach to the characterization of the mechanism of aggregation inhibition was validated highlighting inhibitors mechanism of action and elucidating which are the species whose formation is impeded.

It has been reported that the interaction of the two enantiomers of 9-HSA with the catalytic site of hystone deacetylase, using a molecular modelling approach, is different. The interaction energy of the (R) enantiomer was lower as compared with the (S) indicating a more stable complex formation (N. Calonghi et al 2005). In this work (R) and (S)-9HSA were isolated and their biological activity tested in HT29 cell line. The conclusions that can be drawn from this work can be summarized as follows: (i) Both enantiomers inhibit the enzymatic activity of HDAC1, HDAC2 and HDAC3, being (R) more active; (ii) (R) and (S) inhibitory effect induces an increase in histone H4 acetylation; (iii) the antiproliferative effect induced by (R) is more pronounced as much as the increase of p21 transcription and protein level, while the expression of Cyclin D1 is decreased. It can be hypothesized that the interaction of (R)-9HSA with HDAC1 may induce conformational changes in the enzyme causing loss of its interaction with other proteins, like cyclin D1 that, being released from the complex, can be ubiquitinated, thus losing the interaction with CDK and thereby stopping the cell proliferation.

Two novel families of dual binding site acetylcholinesterase (AChE) inhibitors have been developed, consisting of a tacrine or 6-chlorotacrine unit as the active site interacting moiety, either the 5,6-dimethoxy-2-[(4-piperidinyl)methyl]-1-indanone fragment of donepezil (or the indane derivative thereof) or a 5-phenylpyrano[3,2-c]quinoline system, reminiscent to the tryciclic core of propidium, as the peripheral site interacting unit, and a linker of suitable length as to allow the simultaneous binding at both sites. These hybrid compounds are all potent and selective inhibitors of human AChE, and more interestingly, are able to interfere in vitro both formation and aggregation of the beta-amyloid peptide, the latter effects endowing these compounds with the potential to modify Alzheimer's disease progression.

Advanced nanofabrication is capable of producing structures in the vicinity of the size of large biomolecules or their aggregates. Some of these protein aggregates emerge as having deleterious medical effects, e.g., degenerative diseases, or essential for biological processes, e.g., actin, cytoskeleton formation. Therefore it became possible, and important, to think of ways of interacting nanostructured surfaces with biomolecular aggregates in a designed manner. Along this line of thinking, we report on a preliminary atomic force microscopy (AFM) investigation of the behavior of F-actin on unstructured surfaces (mica, silicon) and nanostructured surface (13 nm height nanostructured silicon surface).

Il capitolo tratta i principi generali e le applicazioni in campo farmaceutico della tecnica separativa elettroforesi capillare. In particolare, sono sviluppati gli aspetti teorici e quelli strumentali assieme alle diverse modalità in cui tale tecnica può essere realizzata.

La maggior parte delle analisi viene effettuata prelevando una parte o porzione del materiale/prodotto da esaminare (formulazione farmaceutica, estratto da pianta medicinale, prodotto cosmetico, etc.) per sottoporlo ad un procedimento di analisi di laboratorio o talvolta anche sul posto. Questa porzione viene detta campione ed è costituita dagli analiti (principi attivi), di cui si vuole verificare la presenz e7o il contenuto e dalla matrice (insieme dei componenti inattivi) che racchiude in forma dispersa gli analiti. L'intero processo di analisi prevede quindi varie fasi, ognuna delle quali richiede la dovuta attenzione. La prima fase è il campionamento cioè il prelievo del campione dal materiale in esame. Di solitp c'è un certo ritardo tra il momento della raccolta del campione e la sua analisi; pertanto la coservazione del campione può costituire un punto critico del processo di lavoro analitico. Infine, la terza importante fase è la preparazione del campione. Questa fase che precede la misura analitica è di estrema importanza, infatti, il campione non puà essere introdotto direttamente tal quale nello strumneto di analisi ma deve essere reso "presentabile" allo strumento analitico.In altre parole preparare il campione significa sottoporre il campione ad un tratatmento appropriato per portatre gli analiti nelle condizioni adatte di solvente, pH, concentrazione, forma molecolare rilevabile ed assenza di interferenze, assicurando nel contempo la loro stabilità ed il loro recupero quantitativo e/o riproducibile dalla matrice in cui si trovano dispersi. La preparzione del campione quindi prevede solitamente un processo di estrazione degli analiti dalla loro matrice mediante l'impiego di gas inerti, solventi, fluidi supercritici o adsorbenti solidi. Le principali tecniche impiegate per estrarre un soluto (principio attivo) dalla matrice di un campione di interesse farmaceutico possono essere riassunte nel modo seguente: (i) estrazioni liquido-solido in cui si cerca di rimuovere il soluto da una matrice solida mediante un solvente o un fluido supercritico, (ii) estrazione liquido-liquido che consente di trasferire in modo selettivo un soluto da una fase liquida ad un'altra fase liquida immiscibile con la prima, (iii) estrazione in fase solida che consente di estrarre un soluto da un campione liquido o gassoso mediante un materiale solido adsorbente sul quale il soluto viene selettivamente concentrato e poi rimosso con quantità minime di solvente o per desorbimento termico.

The aspartic protease beta-secretase (BACE-1) is the rate-limiting enzyme for the generation of the amyloid protein (Aβ), a main and core component of Alzheimer’s disease (AD) senile plaques [1]. Amyloid precursor protein (APP) proteolysis and Aβ generation represent potential targets for AD therapy. Moreover, mice with inactivated genes encoding BACE1 have not showed anatomical or physiological abnormalities [2]. In the last decade, the BACE-1 role in AD pathogenysis was well established and the search for new non peptidic inhibitors has been intensified [3]. BACE-1 activity is usually detected in FRET assays, by using peptidic substrates coupled to donor-acceptor pairs. The in-solution assays have some drawbacks, including the long time assay and the high enzyme consumption. Immobilization of enzymes can be a valid solution to overcome these problems. An immobilized enzyme reactor (IMER) results an interesting tool for the fast screening of enzyme inhibitors. In a previous work, we developed and characterized a hrBACE-1-IMER on an ethylendiamine (EDA) CIM disk by using a bidimensional LC system [4]. Here, to speed and automatise the analysis, a novel LC methods was developed. A fluorogenic peptide was used as BACE-1 substrate; enzymatic activity was evaluated by measuring the peak area of the hydrolysis product eluted in less than 10 min. The specificity of the product formation was confirmed by injecting the substrate on a blank EDA-CIM. The method was validated by measuring enzyme inhibition from standard inhibitors and the results obtained were found in agreement with the data reported in literature, confirming the validity of the hrBACE-1-IMER as a tool for the fast screening of novel inhibitors.

Following our previous in vitro circular dichroism and fluorometric studies on the role of chaperone protein (acetylcholinesterase) in amyloid- peptide (A) aggregation1, and on A direct self assembly2, AFM and MALDI-ToF mass spectrometry approaches are here presented for the characterization of the dimension and molecular weight of A (1-42) aggregates. The results presented here provide nanometric resolution of the main structures characteristic of the several steps from monomeric A (1-42) to mature fibrils in vitro. Oligomeric globular aggregates of A (1-42) precede the appearance of protofibrils, the first fibrillar species. The most abundant form of A (1-42) fibril exhibits a nodular structure with a ~100-nm periodicity. These studies are in the frame of BISNES european project3, focused on the design, fabrication and operation of biomimetic nanostructured surfaces which precisely control the self-assembly of biomolecules in long-range architectures.