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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.

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

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.

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.

A liquid chromatographic method has been applied for the analysis of two antibiotics widely used in the veterinary field, oxytetracycline dihydrate and chlortetracycline hydrochloride in premixes for medicated feeding stuffs for veterinary use. In particular, the validated method was employed to study the releasing profile of each drug from two formulations, a commercially available and a new formulation, having different excipient composition. The dissolution profiles obtained from the chromatographic analysis allowed to verify the in vitro bio-equivalence of the commercial and the new formulations for oxytetracycline and chlortetracycline.

Histone proteins are subject to a range of post-translational modifications in living cells. The combinatorial nature of these modifications reflects in the so called "histone code" that modulates chromatin structure and function during development, growth, differentiation, and homeostasis of cells. Histone acetylation and deacetylation are controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs) enzymes. HDAC inhibitors (HDACi) increase acetylation levels, induce open chromatin structure and increased gene transcription, being found to function as potential chemotherapeutic in tumour cell types (1). Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, it is here reported a LC-MS approach for qualitative and quantitative proteomic analysis of histones extracted from human colon cancer cells after treatment with HDACi. LC-MS-based methods were applied in a quantitative proteomic study of the dose and time response effect of HDACi on histone acetylation in HT29 colon cancer cell cultures. A correlation was established between the degree of histone post-translational modifications and the cell cycle effects caused by treatment of HT29 colon cancer cells with selective and non selective HDACi. This correlation affords a mean to better understand the mechanism of action of new, more potent and selective HDACi directly on the cells. On the basis of these studies, LC-MS-based quantitative proteomic analysis of histone post-translational modifications results as a viable approach for functional analysis of colon cancer candidate drugs, such as HDACi.

We extended the AChE inhibitors SAR study to newly synthesized compounds based on the lead compound xantostigmine. Docking and molecular dynamics simulations were carried out to both define a new computational protocol, and to acquire a better understanding of the SAR data. These computations prompted us to evaluate two of the synthesized compounds as inhibitors of AChE-induced Abeta aggregation.Structure-activity relationship studies on acetylcholinesterase (AChE) inhibitors were extended to newly synthesized compounds derived from the lead compound xantostigmine (1). The xanthone ring of compound 1 was replaced with several different scaffolds based on the benzopyran skeleton, linked to the tertiary amino nitrogen through an heptyloxy chain. These modifications resulted in 19 new compounds, most of them showing activity in the nanomolar-subnanomolar range. Docking and molecular dynamics simulations were carried out to both define a new computational protocol for the simulation of pseudo-irreversibile AChE covalent inhibitors, and to acquire a better understanding of the structure-activity relationships of the present series of compounds. The results of this computational work prompted us to to evaluate the ability of compounds 5 and 13 to inhibit acetylcholinesterase-induced Abeta aggregation.

In the last decade, the design of novel classes of inhibitors of the enzyme acetylcholinesterase (AChE) as therapeutic interventions for Alzheimer’s disease (AD) has been mostly driven by the pivotal finding that AChE can bind the β-amyloid peptide (Aβ), thereby promoting Aβ aggregation as an early event in the neurodegenerative cascade of AD. Blockade of the peripheral site of AChE, the Aβ recognition zone within the enzyme, is expected to affect the AChE-induced Aβ aggregation and could be a potential strategy to modulate the progression of AD. Novel classes of AChE inhibitors (AChEIs) targeting the peripheral site of AChE are emerging as promising disease-modifying anti-Alzheimer drug candidates. Of particular interest are the so-called dual binding site AChEIs, i.e. inhibitors able to simultaneously bind both the active and peripheral sites of AChE, which exhibit significant Aβ antiaggregating effects and high AChE inhibitory potencies. Two isomeric series of dual binding site AChEIs have been designed, synthesized, and tested for their ability to inhibit AChE, butyrylcholinesterase, AChE-induced and self-induced Aβ aggregation and β-secretase (BACE-1). The new hybrids consist of a unit of 6-chlorotacrine and a multicomponent reactionderived pyrano[3,2-c]quinoline scaffold as the active-site and peripheral-site interacting moieties, respectively, connected through an oligomethylene linker containing an amido group at variable position. Molecular modeling studies have confirmed the dual site binding of these hybrids, which retain the potent and selective human AChE inhibitory activity of the parent 6-chlorotacrine, while exhibiting a significant in vitro Aβ anti-aggregating effect and BACE-1 inhibitory activity, thus constituting promising anti-Alzheimerdrug candidates.

A novel series of donepezil-tacrine hybrids designed to simultaneously interact with the active, peripheral and midgorge binding sites of acetylcholinesterase (AChE) have been synthesized and tested for their ability to inhibit AChE, butyrylcholinesterase (BChE), and AChE-induced beta-amyloid (A-beta) aggregation. These compounds consist of a unit of tacrine or 6-chlorotacrine, which occupies the same position than tacrine at the AChE active site, and the 5,6-dimethoxy-2-[(4-piperidinyl)methyl]-1-indanone moiety of donepezil (or the indane derivative thereof), whose position along the enzyme gorge and the peripheral site can be modulated by a suitable tether which connects tacrine and donepezil fragments. All of the new compounds are highly potent inhibitors of bovine and human AChE and BChE, exhibiting IC50 values in the subnanomolar or low nanomolar range in most cases. Moreover, six out of the eight hybrids of the series, particularly those bearing an indane moiety, exhibit a significant A-beta antiaggregating activity, which makes them promising anti-Alzheimer drug candidates.

Novel multi-target-directed ligands were designed by replacing the inner dipiperidino function of 3 with less flexible or completely rigid moieties to obtain compounds endowed with multiple biological properties that might be relevant to Alzheimer's disease. 15 was the most interesting, inhibiting AChE in the nanomolar range and inhibiting AChE-induced and self-promoted beta-amyloid aggregation in the micromolar range.

Trypsin is the most used proteolytic enzyme for protein digestion and Peptide Mass Fingerprinting (PMF). Digestions can be performed in solution with classical incubations protocols of 4-24 h. However, in the search for faster and automated methodologies, an interesting alternative is the use of solid phase digestion achieved by immobilizing trypsin on methacrylate-based monolithic CIM (Convective Interaction Media®) disks. Rapid on-line digestion (5 min) of numerous proteins was achieved by inserting the trypsin-based IMER (Immobilized Enzyme Reactor) in a liquid chromatography system hyphenated to (or with?) tandem mass spectrometry (IMER-LC-MS/MS). On-line results were compared in terms of sequence coverage with classical in-solution digestions and commercially available Poroszyme® cartridge. Presenting high performances in terms of digestion time, long term stability and costs, the monolithic IMER demonstrated to be an attractive tool to perfor rapid and automated protein digestions. One of the main reasons for new chemical entities failure in drug discovery could be attributed to inadequate pharmacokinetic properties. Therefore, the determination of major metabolic pathways and the study of drug-drug interactions are mandatory for optimizing suitable drug candidates. In this context, ethylenediamine (EDA) CIM® minidisks were selected for non-covalent immobilization of recombinant-expressed CYP2D6 and CYP3A4 liposomes (RECO System®). Resulting CYP450-based IMERs were integrated in a fully automated IMER-LC-MS/MS for on-line phase I drug metabolism studies with probe substrates and a known competitive inhibitor. Results showed that the optimized immobilization procedure was suitable to maintain the enzyme activity and that the inclusion of CYPs in the IMER significantly

It is here reported the preparation and comparison of different monolithic micro-IMERs (Immobilized Enzyme Reactors) containing covalently immobilized human recombinant acetylcholinesterase (AChE). In particular, following previous studies on AChE-IMERs (1,2), object of this work was the covalent immobilisation of the target enzyme on disk-shaped columns of smaller dimensions (6 x 2 mm, Bed Volume = 56 mL) to increase the enzyme density on the matrix surface. Since the surface chemistry of the chosen chromatographic support will influence the yield of immobilisation as well as the environment in which the target enzyme will be inserted, the choice of an appropriate support for IMERs development represents an important issue. Moreover, “aspecific” interactions between matrix and substrate and inhibitors will also depend on the chosen material. In this context, CIM disks with different chemistries were chosen for AChE immobilisation, in particular, ethylendiamino (EDA), epoxy and CDI-CIM mini disks were selected. Moreover the introduction of an appropriate spacer arm was evaluated when required. On-line automated HPLC inhibition studies on a selected series of AChE inhibitors were performed; Aspecific interactions were evaluated and results obtained for the same compound on different CIM-based IMERs were compared. Results showed that purposely pre-derivatized CDI-based IMER gave promising eluting profile and low aspecific interactions and may therefore represent a valid chromatographic support for the development of monolithic micro-IMERs. (1) M. Bartolini, V. Cavrini, V. Andrisano: Monolithic micro-immobilized-enzyme reactor with human recombinant acetylcholinesterase for on-line inhibition studies. J Chromatogr A 2004, 1031: 27-34. (2) M. Bartolini, V. Cavrini, V. Andrisano: Choosing the right chromatographic support in making a new acetylcholinesterase-micro-immobilised enzyme reactor for drug discovery. J Chromatogr A. 2005, 1065:135-44.

During the last decade new combinatorial techniques have been developed that allow for synthesis of vast quantities of potential therapeutic compounds. At the same time, in parallel with the synthetic process, analytical techniques must be capable of screening a large number of compounds in a high-throughput manner with precision and accuracy. In the last few years, immobilised enzyme reactors (IMERs) on monolithic matrices have been developed by numerous groups and have proven to be a useful and economic alternative to conventional in-solution methods, when automation and fast data output are required. In particular, considering the high cost and difficulty in over-expression, isolation and purification of recombinant enzymes, this analytical technique represents an extremely useful approach to preserve the activity of the small amount of enzyme available. In the IMER format, enzymes are ready to be reused and can be coupled to chromatographic systems and appropriate detectors. This coupling generally increases automation, reproducibility and analyses accuracy and reduces sample handling and operator time consumption. Monoliths are considered a novel generation of stationary phases whose special feature is the fast separation and enzymatic conversion due to lack of diffusion resistance during mass transfer. The large pores of monolithic materials allow high-speed analysis and low back pressure. Preliminary experiments carried out with enzymes immobilized onto compact porous supports have shown that such enzyme reactors have much higher rates of conversion than the reactors in which enzymes have been immobilized onto bulk porous supports. In the field of drug discovery, monolithic IMERs can be reliably applied to rapidly screen for potential drugs candidates to lead selection, to perform potent inhibitor’s kinetic studies, and for ADMET parameters determination, an interesting application being for the early estimates of the drug metabolism, toxicity and possible drug-drug interactions. Here, a few IMERs applications will be presented, including cholinesterase and BACE-1 (beta secretase) immobilized reactors, which were validated for the determination of the mechanism of action and inhibitory constants of new leads for the treatment of Alzheimer’s disease in a highly reliable and automated mode. Moreover, the preparation of monolithic disk-shaped mini-columns (2 mm x 6 mm I.D.) containing immobilized cytochrome P450 as tools for drug metabolim studies, will be described.

A novel series of donepezil-tacrine hybrids designed to simultaneously interact with the active, peripheral and midgorge binding sites of acetylcholinesterase (AChE) have been synthesized and tested for their ability to inhibit AChE, butyrylcholinesterase (BChE), and AChE-induced A beta aggregation. These compounds consist of a unit of tacrine or 6-chlorotacrine, which occupies the same position as tacrine at the AChE active site, and the 5,6-dimethoxy-2-[(4-piperidinyl)methyl]-1-indanone moiety of donepezil (or the indane derivative thereof), whose position along the enzyme gorge and the peripheral site can be modulated by a suitable tether that connects tacrine and donepezil fragments. All of the new compounds are highly potent inhibitors of bovine and human AChE and BChE, exhibiting IC50 values in the subnanomolar or low nanomolar range in most cases. Moreover, six out of the eight hybrids of the series, particularly those bearing an indane moiety, exhibit a significant A beta antiaggregating activity, which makes them promising anti-Alzheimer drug candidates.

The complex etiology of Alzheimer's disease (AD) prompts scientists to develop multitarget strategies to combat causes and symptoms. We therefore designed, synthesized, and tested new hybrid molecules linking a benzofuran ring to a N-methyl- N-benzylamine through a heptyloxy chain, affording a series of potential multifunctional drugs for AD. The cholinesterase inhibitory activity was extended to the inhibition of Abeta fibril formation for 1, 3, and 5. Compound 3 showed an additional neuroprotective effect.

In the search for new therapeutics for Alzheimer’s Disease (AD), the investigation of the mechanism of action of new cholinesterase inhibitors represents a key aspect for new lead selection and subsequent optimization and development. However, the in vitro characterization of drug candidates on isolated target enzymes often requires a long operating time and a large amount of disposable expensive material. The recent development and aplication of stable immobilized enzyme reactors (IMERs) can ameliorate these concerns, and such in-solution methods should be potentially utilized in automated procedures to obtain efficient and effective higher throughput screening. In this context, the focus of this work was the development and characterization of a new IMER containing immobilized human recombinant butyrylcholinesterase (BChE) for the on-line kinetic characterization of specific, pseudo-irreversible and brain-targeted BChE inhibitors as potential drug candidates for AD. The selected phenserine and cymserine analogues were synthesized, characterized by classical ex vivo binding assays [1], and assessed by BChE-IMER. For a pseudo-irreversible inhibitor, the kinetics constants describe the mode and duration of enzyme inhibition that will, in turn, influence the duration of the inhibitors’s in vivo pharmacological actions. The kinetic constants were determined for selected phenserine and cymserine analogues using a purposely-designed on-line procedure. Specifically, agents were inserted on to the BChE-IMER that fed directly into a HPLC system connected to a UV-Vis detector, BChE-IMER activity was determined on the basis of the Ellman reaction [2]. The carbamoylation and decarbamoylation phases at different inhibitor concentrations were then monitored continuously over time. Results allowed elucidation of the inhibition duration, mode of action and structure-activity relationships of the inhibitors of interest, which were compared to available values deriving from classical assessment. (1) N.H. Greig, T. Utsuki, D.K. Ingram, Y. Wang, G. Pepeu, C. Scali, Q.S. Yu, I. Mamczarz, H.W. Holloway, T. Giordano, D. Chen, K. Furukawa, K. Sambamurti, A. Brossi, D.K. Lahiri: Selective butyrylcholinesterase inhibition elevates brain acetylcholine, augments learning and lowers Alzheimer beta-amyloid peptide in rodent. Proc Natl Acad Sci USA 2005, 102: 17213-18. (2) G.L. Ellman, K.D. Courtney, V. Jr. Andres, R.M. Featherstone. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961, 7: 88-95.

Alzheimer’s Disease (AD) is associated with a progressive decline in the neurotransmitter acetylcholine (ACh) in the forebrain of afflicted subjects. In healthy human brain, butyrylcholinesterase (BChE, EC 3.1.1.8) has prevalently a supportive role in the hydrolysis of ACh, whereas acetylcholinesterase (AChE) activity predominates. Nevertheless in AD brain, since AChE levels rapidly decrease, the role of BChE rises. It has been demostrated that selectively inhibiting the action of BChE in the brain results in an increase of cerebral acetylcholine levels and improves learning in rats (1). These findings support BChE as key target in AD and may translate into new therapeutics for AD. The goal of the present work is the development and characterization of a new enzyme reactor containing immobilized human recombinant BChE (hrBChE) for the on-line evaluation of the kinetics of new specific, pseudo-irreversible and brain-targeted BChE inhibitors developed by N. Greig and coworkers. For this purpose, human recombinant BChE was covalently immobilized onto an EDA CIM® disk and on-line studies were performed by inserting the IMER into a HPLC system. A specific chromogenic reagent coupled with UV detection was used to follow product formation and to determine both activity and inhibition. Carbamoylation and decarbamoylation constants were determined for a series of physostigmine analogues. Results allowed us to elucidate inhibition duration and structure activity relationships. Moreover, both kinetic constants could be determined in a single experiment. (1)Nigel H. Greig et al. PNAS 2005, 102:17213-8

Memoquin, a recently discovered new drug candidate for the treatment of Alzheimer's disease (AD), is a compound able to interfere with different key targets of AD neurodegeneration. In the present work, the electroactivity of Memoquin, due to the presence of a quinone ring in the molecule, was exploited for the development of a sensitive and selective HPLC chromatographic method with coulometric detection system. For this purpose, a monolithic silica-based stationary phase (Chromolith C18) was coupled with ESA detector and under high flow rate condition (2 mL/min) gave an efficient coulometric detection without a significant backpressure. The monolithic stationary phase and the electrochemical detection were combined to obtain a very fast (less than 7 min) and sensitive analysis of the compound of interest. For this reason, the method was found suitable to determine Memoquin with very high sensitivity (LOD, 0.005 microg/mL; LOQ, 0.016 microg/mL), better than with UV and fluorescence detections, and selectivity resulting potentially suitable for its analysis in biological samples. Moreover, the HPLC method was employed for the separation of Memoquin from other quinone analogs (endowed with different substituent on the quinone ring and length of the lateral chain) and allowed the comparison of the electrochemical properties of the compounds of the series. In the optimized chromatographic conditions it was possible to separate each quinone and to evaluate the influence of the substituent of the quinone ring on the electrochemical properties of the molecule.