Publications by authors named "Marina Naldi"

49 Publications

Determination of effective albumin in patients with decompensated cirrhosis: clinical and prognostic implications.

Hepatology 2021 Mar 12. Epub 2021 Mar 12.

IRCSS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy.

Background & Aims: Circulating albumin in cirrhosis can be dysfunctional due to accumulating structural damages, leading to the concept of effective albumin concentration (eAlb) referring to the albumin portion presenting structural and functional integrity. We aimed to estimate eAlb in patients with decompensated cirrhosis and analyze its relationships with albumin function and clinical outcomes as compared to total albumin concentration (tAlb).

Approach & Results: We evaluated 319 cirrhotic patients hospitalized for acute decompensation (AD) with and without acute-on-chronic liver failure (ACLF) and 18 age- and sex-comparable outpatients with compensated cirrhosis. tAlb was quantified by standard assay, while eAlb was estimated combining liquid chromatography-electrospray ionization-mass spectrometry and standard methods. Albumin binding and detoxification efficiency were evaluated by electron paramagnetic resonance analysis. Circulating albumin in patients with decompensated cirrhosis displayed multiple structural abnormalities, with reversible oxidation and glycation being the most frequent. As a result, eAlb progressively declined with the worsening of cirrhosis, and was superior to tAlb in stratifying patients between compensated cirrhosis, AD and ACLF, as well as patients with and without complications. Moreover, eALB, but not tAlb, was closely associated with binding capacities in ACLF. Finally, eALB at admission predicted the occurrence of ACLF within 30 days and mortality at 90 days better than tAlb.

Conclusion: This large observational study provides the evidence in patients with decompensated cirrhosis that eAlb can be quantified and differentiated from tAlb routinely measured in clinical practice. As compared to tAlb, eAlb is more closely associated with disease severity and albumin dysfunction, and carries a greater prognostic power. These results prompt future research assessing eAlb as a novel biomarker for predicting prognosis and treatment response.
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http://dx.doi.org/10.1002/hep.31798DOI Listing
March 2021

From Combinations to Single-Molecule Polypharmacology-Cromolyn-Ibuprofen Conjugates for Alzheimer's Disease.

Molecules 2021 Feb 19;26(4). Epub 2021 Feb 19.

Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Via Belmeloro 6/Via Selmi 3, 40126 Bologna, Italy.

Despite Alzheimer's disease (AD) incidence being projected to increase worldwide, the drugs currently on the market can only mitigate symptoms. Considering the failures of the classical paradigm "one target-one drug-one disease" in delivering effective medications for AD, polypharmacology appears to be a most viable therapeutic strategy. Polypharmacology can involve combinations of multiple drugs and/or single chemical entities modulating multiple targets. Taking inspiration from an ongoing clinical trial, this work aims to convert a promising cromolyn-ibuprofen drug combination into single-molecule "codrugs." Such codrugs should be able to similarly modulate neuroinflammatory and amyloid pathways, while showing peculiar pros and cons. By exploiting a linking strategy, we designed and synthesized a small set of cromolyn-ibuprofen conjugates (-). Preliminary plasma stability and neurotoxicity assays allowed us to select diamide and ethanolamide as promising compounds for further studies. We investigated their immunomodulatory profile in immortalized microglia cells, in vitro anti-aggregating activity towards Aβ-amyloid self-aggregation, and their cellular neuroprotective effect against Aβ-induced neurotoxicity. The fact that effectively reduced Aβ-induced neuronal death, prompted its investigation into an in vivo model. Notably, was demonstrated to significantly increase the longevity of Aβ-expressing and to improve fly locomotor performance.
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http://dx.doi.org/10.3390/molecules26041112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923232PMC
February 2021

Discovery of an Allosteric Ligand Binding Site in SMYD3 Lysine Methyltransferase.

Chembiochem 2021 Jan 5. Epub 2021 Jan 5.

Department of Chemistry-BMC, Uppsala University, Husargatan 3, 754 24, Uppsala, Sweden.

SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened in a biosensor-based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (K =42 and 84 μM, respectively). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3-HSP90 binding was confirmed (K =13 μM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action.
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http://dx.doi.org/10.1002/cbic.202000736DOI Listing
January 2021

Indole Derivative Interacts with Estrogen Receptor Beta and Inhibits Human Ovarian Cancer Cell Growth.

Molecules 2020 Sep 27;25(19). Epub 2020 Sep 27.

Department of Pharmacy and Biotechnology, University of Bologna, 40121 Bologna, Italy.

Ovarian cancer remains the leading cause of mortality among gynecological tumors. Estrogen receptor beta (ERβ) expression has been suggested to act as a tumor suppressor in epithelial ovarian cancer by reducing both tumor growth and metastasis. ERβ expression abnormalities represent a critical step in the development and progression of ovarian cancer: for these reasons, its re-expression by genetic engineering, as well as the use of targeted ERβ therapies, still constitute an important therapeutic approach. 3-{[2-chloro-1-(4-chlorobenzyl)-5-methoxy-6-methyl-1-indol-3-yl]methylene}-5-hydroxy-6-methyl-1,3-dihydro-2-indol-2-one, referred to here as compound , has been shown to have cytostatic as well cytotoxic effects on various hormone-dependent cancer cell lines. However, the mechanism of its anti-carcinogenic activity is not well understood. Here, we offer a possible explanation of such an effect in the human ovarian cancer cell line IGROV1. Chromatin binding protein assay and liquid chromatography mass spectrometry were exploited to localize and quantify compound in cells. Molecular docking was used to prove compound binding to ERβ. Mass spectrometry-based approaches were used to analyze histone post-translational modifications. Finally, gene expression analyses revealed a set of genes regulated by the ERβ/3 complex, namely CCND1, MYC, CDKN2A, and ESR2, providing possible molecular mechanisms that underline the observed antiproliferative effects.
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http://dx.doi.org/10.3390/molecules25194438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582771PMC
September 2020

Unveiling the molecular mechanisms underpinning biorecognition of early-glycated human serum albumin and receptor for advanced glycation end products.

Anal Bioanal Chem 2020 Jul 4;412(18):4245-4259. Epub 2020 May 4.

Department of Pharmacy and Biotechnology, Alma Mater Studiorum - University of Bologna, Via Belmeloro 6, 40126, Bologna, Italy.

Serum levels of early-glycated albumin are significantly increased in patients with diabetes mellitus and may play a role in worsening inflammatory status and sustaining diabetes-related complications. To investigate possible pathological recognition involving early-glycated albumin and the receptor for advanced glycation end products (RAGE), an early-glycated human serum albumin (HSAgly), with a glycation pattern representative of the glycated HSA form abundant in diabetic patients, and the recombinant human RAGE ectodomain (VC1) were used. Biorecognition between the two interactants was investigated by combining surface plasmon resonance (SPR) analysis and affinity chromatography coupled with mass spectrometry (affinity-MS) for peptide extraction and identification. SPR analysis proved early-glycated albumin could interact with the RAGE ectodomain with a steady-state affinity constant of 6.05 ± 0.96 × 10 M. Such interaction was shown to be specific, as confirmed by a displacement assay with chondroitin sulfate, a known RAGE binder. Affinity-MS studies were performed to map the surface area involved in the recognition. These studies highlighted that a region surrounding Lys525 and part of subdomain IA were involved in VC1 recognition. Finally, an in silico analysis highlighted (i) a key role for glycation at Lys525 (the most commonly glycated residue in HSA in diabetic patients) through a triggering mechanism similar to that previously observed for AGEs or advanced lipoxidation end products and (ii) a stabilizing role for subdomain IA. Albeit a moderate affinity for complex formation, the high plasma levels of early-glycated albumin and high percentage of glycation at Lys525 in diabetic patients make this interaction of possible pathological relevance. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-020-02674-wDOI Listing
July 2020

Functional characterization of multifunctional ligands targeting acetylcholinesterase and alpha 7 nicotinic acetylcholine receptor.

Biochem Pharmacol 2020 07 30;177:114010. Epub 2020 Apr 30.

Normandie Univ, UNIROUEN, INSA Rouen, CNRS, COBRA (UMR 6014), 76000 Rouen, France. Electronic address:

Alzheimer's disease (AD) is a neurodegenerative disorder associated with cholinergic dysfunction, provoking memory loss and cognitive dysfunction in elderly patients. The cholinergic hypothesis provided over the years with molecular targets for developing palliative treatments for AD, acting on the cholinergic system, namely, acetylcholinesterase and α7 nicotinic acetylcholine receptor (α7 nAChR). In our synthetic work, we used "click-chemistry" to synthesize two Multi Target Directed Ligands (MTDLs) MB105 and MB118 carrying tacrine and quinuclidine scaffolds which are known for their anticholinesterase and α7 nAChR agonist activities, respectively. Both, MB105 and MB118, inhibit human acetylcholinesterase and human butyrylcholinesterase in the nanomolar range. Electrophysiological recordings on Xenopus laevis oocytes expressing human α7 nAChR showed that MB105 and MB118 acted as partial agonists of the referred nicotinic receptor, albeit, with different potencies despite their similar structure. The different substitution at C-3 on the 2,3-disubstituted quinuclidine scaffold may account for the significantly lower potency of MB118 compared to MB105. Electrophysiological recordings also showed that the tacrine precursor MB320 behaved as a competitive antagonist of human α7 nAChR, in the micromolar range, while the quinuclidine synthetic precursor MB099 acted as a partial agonist. Taken all together, MB105 behaved as a partial agonist of α7 nAChR at concentrations where it completely inhibited human acetylcholinesterase activity paving the way for the design of novel MTDLs for palliative treatment of AD.
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http://dx.doi.org/10.1016/j.bcp.2020.114010DOI Listing
July 2020

In vitro thrombogenicity of drug-eluting and bare metal stents.

Thromb Res 2020 01 14;185:43-48. Epub 2019 Nov 14.

Columbia University Medical Center/New York-Presbyterian Hospital and the Cardiovascular Research Foundation, New York, NY, United States.

Aims: We sought to investigate the thrombogenicity of different DES and BMS in an in vitro system of stent perfusion.

Material And Methods: The experimental model consisted of a peristaltic pump connected to 4 parallel silicone tubes in which different stents were deployed. Blood was drawn from healthy volunteers and the amount of stent surfaced-induced thrombus deposition was determined using I-fibrinogen.

Results: Compared to Resolute, Biomatrix and Vision, Xience was associated with the lowest amount of stent surface-induced thrombus formation, with a significant difference compared to Vision (I-fibrinogen median value deposition [IQ range]: 50 ng [25-98] versus 560 ng [320-1520], respectively, p < 0.05), but not to other DES. In the second set of experiments Fluoropolymer-coated BMS not eluting drug was associated with a significant 3-fold reduction in I-fibrinogen deposition (245 ng [80-300]) compared to Vision (625 ng [320-760], p < 0.05), but a 7-fold increase compared to Xience (35 ng [20-60], p < 0.05). Finally Xience was associated with a significantly greater absorption of albumin compared to BMS.

Conclusions: In an in vitro system of stent perfusion, Xience was associated with the lowest amount of stent surface-induced thrombus formation compared with Resolute, Biomatrix and Vision, with a noted synergistic effect between the fluoropolymer and the drug.
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http://dx.doi.org/10.1016/j.thromres.2019.11.016DOI Listing
January 2020

Dual-Acting Cholinesterase-Human Cannabinoid Receptor 2 Ligands Show Pronounced Neuroprotection in Vitro and Overadditive and Disease-Modifying Neuroprotective Effects in Vivo.

J Med Chem 2019 10 14;62(20):9078-9102. Epub 2019 Oct 14.

Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry , Julius Maximilian University of Würzburg , Am Hubland , 97074 Würzburg , Germany.

We have designed and synthesized a series of 14 hybrid molecules out of the cholinesterase (ChE) inhibitor tacrine and a benzimidazole-based human cannabinoid receptor subtype 2 (hCBR) agonist and investigated them in vitro and in vivo. The compounds are potent ChE inhibitors, and for the most promising hybrids, the mechanism of human acetylcholinesterase (hAChE) inhibition as well as their ability to interfere with AChE-induced aggregation of β-amyloid (Aβ), and Aβ self-aggregation was assessed. All hybrids were evaluated for affinity and selectivity for hCBR and hCBR. To ensure that the hybrids retained their agonist character, the expression of cAMP-regulated genes was quantified, and potency and efficacy were determined. Additionally, the effects of the hybrids on microglia activation and neuroprotection on HT-22 cells were investigated. The most promising in vitro hybrids showed pronounced neuroprotection in an Alzheimer's mouse model at low dosage (0.1 mg/kg, i.p.), lacking hepatotoxicity even at high dose (3 mg/kg, i.p.).
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http://dx.doi.org/10.1021/acs.jmedchem.9b00623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640639PMC
October 2019

Advanced analytical methodologies in Alzheimer's disease drug discovery.

J Pharm Biomed Anal 2020 Jan 29;178:112899. Epub 2019 Sep 29.

Department for Life Quality Studies, Alma Mater Studiorum - University of Bologna, Rimini, Italy. Electronic address:

Despite the constant progress in the understanding of the etiopathogenesis of Alzheimer's disease (AD) over the last 50 years, just four long-standing drugs are currently used for AD therapy. This article reviews the analytical methodologies developed and applied in the last five years to address the early-stage tasks of the AD drug discovery process: the fast selection of active compounds (hits) and the comprehension of the ligand binding mechanism of the compound chosen to be the lead in the forthcoming development. The reviewed analytical methodologies face the most investigated pharmacological protein targets (amyloids, secretases, kinases, cholinesterases) and specific receptor- and enzyme-mediated effects in neurotransmission, neuroprotection and neurodegeneration. Some of these methodologies are noteworthy for their use in middle/high-throughput screening campaigns during hit selection (e.g. surface plasmon resonance biosensing, fluorescence resonance energy transfer assays), whereas some others (circular dichroism and nuclear magnetic resonance spectroscopies, ion mobility-mass spectrometry) can provide in-depth information about the structure, conformation and ligand binding properties of target proteins.
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http://dx.doi.org/10.1016/j.jpba.2019.112899DOI Listing
January 2020

Investigating in Vitro Amyloid Peptide 1-42 Aggregation: Impact of Higher Molecular Weight Stable Adducts.

ACS Omega 2019 Jul 18;4(7):12308-12318. Epub 2019 Jul 18.

Department for Life Quality Studies, Alma Mater Studiorum Università di Bologna, Rimini 47921, Italy.

The self-assembly of amyloid peptides (Aβ), in particular Aβ, into oligomers and fibrils is one of the main pathological events related to Alzheimer's disease. Recent studies have demonstrated the ability of carbon monoxide-releasing molecules (CORMs) to protect neurons and astrocytes from Aβ toxicity. In fact, CORMs are able to carry and release controlled levels of CO and are known to exert a wide range of anti-inflammatory and anti-apoptotic activities at physiologically relevant concentrations. In order to investigate the direct effects of CORMs on Aβ, we studied the reactivity of CORM-2 and CORM-3 with Aβ and the potential inhibition of its aggregation by mass spectrometry (MS), as well as fluorescence and circular dichroism spectroscopies. The application of an electrospray ionization-MS (ESI-MS) method allowed the detection of stable Aβ/CORMs adducts, involving the addition of the Ru(CO) portion of CORMs at histidine residues on the Aβ skeleton. Moreover, CORMs showed anti-aggregating properties through formation of stable adducts with Aβ as demonstrated by a thioflavin T fluorescence assay and MS analysis. As further proof, comparison of the CD spectra of Aβ recorded in the absence and in the presence of CORM-3 at a 1:1 molar ratio showed the ability of CORM-3 to stabilize the peptide in its soluble, unordered conformation, thereby preventing its misfolding and aggregation. This multi-methodological investigation revealed novel interactions between Aβ and CORMs, contributing new insights into the proposed neuroprotective mechanisms mediated by CORMs and disclosing a new strategy to divert amyloid aggregation and toxicity.
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http://dx.doi.org/10.1021/acsomega.9b01531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682006PMC
July 2019

Unveiling the Biochemistry of the Epigenetic Regulator SMYD3.

Biochemistry 2019 09 21;58(35):3634-3645. Epub 2019 Aug 21.

Department of Chemistry - BMC , Uppsala University , Uppsala , Sweden.

SET and MYND domain-containing protein 3 (SMYD3) is a lysine methyltransferase that plays a central role in a variety of cancer diseases, exerting its pro-oncogenic activity by methylation of key proteins, of both nuclear and cytoplasmic nature. However, the role of SMYD3 in the initiation and progression of cancer is not yet fully understood and further biochemical characterization is required to support the discovery of therapeutics targeting this enzyme. We have therefore developed robust protocols for production, handling, and crystallization of SMYD3 and biophysical and biochemical assays for clarification of SMYD3 biochemistry and identification of useful lead compounds. Specifically, a time-resolved biosensor assay was developed for kinetic characterization of SMYD3 interactions. Functional differences in SMYD3 interactions with its natural small molecule ligands SAM and SAH were revealed, with SAM forming a very stable complex. A variety of peptides mimicking putative substrates of SMYD3 were explored in order to expose structural features important for recognition. The interaction between SMYD3 and some peptides was influenced by SAM. A nonradioactive SMYD3 activity assay using liquid chromatography-mass spectrometry (LC-MS) analysis explored substrate features of importance also for methylation. Methylation was notable only toward MAP kinase kinase kinase 2 (MAP3K2_K)-mimicking peptides, although binary and tertiary complexes were detected also with other peptides. The analysis supported a random bi-bi mechanistic model for SMYD3 methyltransferase catalysis. Our work unveiled complexities in SMYD3 biochemistry and resulted in procedures suitable for further studies and identification of novel starting points for design of effective and specific leads for this potential oncology target.
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http://dx.doi.org/10.1021/acs.biochem.9b00420DOI Listing
September 2019

Tacrine-O-protected phenolics heterodimers as multitarget-directed ligands against Alzheimer's disease: Selective subnanomolar BuChE inhibitors.

Eur J Med Chem 2019 Nov 22;181:111550. Epub 2019 Jul 22.

Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado 1203, E-41071, Sevilla, Spain. Electronic address:

Concerned by the devastating effects of Alzheimer's disease, and the lack of effective drugs, we have carried out the design of a series of tacrine-phenolic heterodimers in order to tackle the multifactorial nature of the disease. Hybridization of both pharmacophores involved the modification of the nature (imino, amino, ether) and the length of the tether, together with the type (hydroxy, methoxy, benzyloxy), number and position of the substituents on the aromatic residue. Title compounds were found to be strong and selective inhibitors of human BuChE (from low nanomolar to subnanomolar range), an enzyme that becomes crucial in the more advanced stages of the disease. The lead compound, bearing an ether-type tether, had an IC50 value of 0.52 nM against human BuChE, and a selectivity index of 323, with an 85-fold increase of activity compared to parent tacrine; key interactions were analysed using molecular modelling. Moreover, it also inhibited the self-aggregation of Aβ42, lacking neurotoxicity up to 5 μM concentration, and showed neuroprotective activity in primary rat neurons in a serum and K+ deprivation model, widely employed for reproducing neuronal injury and senescence. Moreover, low hepatoxicity effects and complete stability under physiological conditions were found for that compound. So, overall, our lead compound can be considered as a promising multitarget-directed ligand against Alzheimer's disease, and a good candidate for developing new drugs.
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http://dx.doi.org/10.1016/j.ejmech.2019.07.053DOI Listing
November 2019

Discovery of novel benzofuran-based compounds with neuroprotective and immunomodulatory properties for Alzheimer's disease treatment.

Eur J Med Chem 2019 Sep 31;178:243-258. Epub 2019 May 31.

Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Via Belmeloro 6, 40126, Bologna, Italy. Electronic address:

To address the multifactorial nature of Alzheimer's Disease (AD), a multi-target-directed ligand approach was herein developed. As a follow-up of our previous studies, a small library of newly designed 2-arylbenzofuran derivatives was evaluated towards cholinesterases and cannabinoid receptors. The two most promising compounds, 8 and 10, were then assessed for their neuroprotective activity and for their ability to modulate the microglial phenotype. Compound 8 emerged as able to fight AD from several directions: it restored the cholinergic system by inhibiting butyrylcholinesterase, showed neuroprotective activity against Aβ oligomers, was a potent and selective CB ligand and had immunomodulatory effects, switching microglia from the pro-inflammatory M1 to the neuroprotective M2 phenotype. Derivative 10 was a potent CB inverse agonist with promising immunomodulatory properties and could be considered as a tool for investigating the role of CB receptors and for developing potential immunomodulating drugs addressing the endocannabinoid system.
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http://dx.doi.org/10.1016/j.ejmech.2019.05.080DOI Listing
September 2019

Polycyclic Maleimide-based Scaffold as New Privileged Structure for Navigating the Cannabinoid System Opportunities.

ACS Med Chem Lett 2019 Apr 26;10(4):596-600. Epub 2019 Feb 26.

Department of Pharmacy and Biotechnology, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy.

The discovery of the relevant role played by a dysregulation of the endogenous cannabinoid system in several pathological conditions has prompted an extensive research in this field. In this Letter, a series of cannabinoid receptor ligands bearing a previously unexplored polycyclic scaffold was designed and synthesized, in order to evaluate the potential of a new easily affordable privileged structure. The new compounds showed an appreciable affinity and a significant selectivity for the CB2 receptor and are endowed with an intriguing noncompetitive antagonist behavior. Due to the ability of the polycyclic structure to be easily modified in different ways, these compounds could represent convenient chemical tools to be exploited in order to better understand the endocannabinoid system impact on physiopathological conditions.
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http://dx.doi.org/10.1021/acsmedchemlett.8b00594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466824PMC
April 2019

Design, biological evaluation and X-ray crystallography of nanomolar multifunctional ligands targeting simultaneously acetylcholinesterase and glycogen synthase kinase-3.

Eur J Med Chem 2019 Apr 13;168:58-77. Epub 2019 Feb 13.

Normandie Univ, UNIROUEN, INSA Rouen, CNRS, COBRA (UMR 6014), 76000, Rouen, France. Electronic address:

Both cholinesterases (AChE and BChE) and kinases, such as GSK-3α/β, are associated with the pathology of Alzheimer's disease. Two scaffolds, targeting AChE (tacrine) and GSK-3α/β (valmerin) simultaneously, were assembled, using copper(I)-catalysed azide alkyne cycloaddition (CuAAC), to generate a new series of multifunctional ligands. A series of eight multi-target directed ligands (MTDLs) was synthesized and evaluated in vitro and in cell cultures. Molecular docking studies, together with the crystal structures of three MTDL/TcAChE complexes, with three tacrine-valmerin hybrids allowed designing an appropriate linker containing a 1,2,3-triazole moiety whose incorporation preserved, and even increased, the original inhibitory potencies of the two selected pharmacophores toward the two targets. Most of the new derivatives exhibited nanomolar affinity for both targets, and the most potent compound of the series displayed inhibitory potencies of 9.5 nM for human acetylcholinesterase (hAChE) and 7 nM for GSK-3α/β. These novel dual MTDLs may serve as suitable leads for further development, since, in the micromolar range, they exhibited low cytotoxicity on a panel of representative human cell lines including the human neuroblastoma cell line SH-SY5Y. Moreover, these tacrine-valmerin hybrids displayed a good ability to penetrate the blood-brain barrier (BBB) without interacting with efflux pumps such as P-gp.
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http://dx.doi.org/10.1016/j.ejmech.2018.12.063DOI Listing
April 2019

New insights into the altered binding capacity of pharmaceutical-grade human serum albumin: site-specific binding studies by induced circular dichroism spectroscopy.

J Pharm Biomed Anal 2019 Jan 12;162:171-178. Epub 2018 Sep 12.

Department of Pharmacy and Biotechnology, Alma Mater Studiorum - University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy. Electronic address:

The ADMET profile of drugs is strongly affected by human serum albumin (HSA), due to its leading role as carrier of poorly soluble compounds in plasma; a critical assessment of the binding capacity of HSA and the evaluation of binding competition between drugs are therefore pivotal for a reliable pharmacokinetic and pharmacodynamic characterization. In clinical practice, a potential source of impairment in the binding properties of HSA is the use of octanoate and N-acetyltryptophan as stabilizers during the production of pharmaceutical-grade HSA for infusion (i-HSA), which is currently administered in the treatment of a growing range of pathological conditions. The peculiar sensitivity of circular dichroism (CD) spectroscopy towards the stereochemical features of high-affinity binding events is herein exploited to achieve a site-specific assessment of the effect of stabilizers on the binding properties of i-HSA. The binding affinity and capacity of fatty-acid-free HSA towards site-selective induced circular dichroism (ICD) markers for the three high-affinity binding sites of HSA was compared to that of i-HSA submitted to ultrafiltration and dialysis to remove both stabilizers. Results showed a considerable impairment of the binding capacity of i-HSA at site II and a relatively lower influence on the binding properties of site I. Ultrafiltration proved to be ineffective in depleting octanoate, while the proposed dialysis protocol, which involves a pH-induced reversible unfolding of the protein, resulted in a total clearance of both stabilizers, confirmed by the full restoration of the binding properties of HSA at all binding sites. The outcomes of this study proved that CD spectroscopy is a suitable technique to evaluate the binding properties of i-HSA, ensuring an assessment of the availability of the binding sites and the possibility of monitoring the clearance of stabilizers. Eventually, the proposed method for their depletion might constitute a connection bridge between albumin in vitro studies and its clinical applications.
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http://dx.doi.org/10.1016/j.jpba.2018.09.022DOI Listing
January 2019

Immobilized enzyme-based analytical tools in the -omics era: Recent advances.

J Pharm Biomed Anal 2018 Oct 31;160:222-237. Epub 2018 Jul 31.

Department of Pharmacy and Biotechnology, Alma Mater Studiorum - University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy. Electronic address:

Protein analysis is a field under rapid development mainly thanks to technological advances which have granted miniaturization of analytical devices, automation and higher detection sensitivity. The interest in the field has paralleled the expansion of the -omics era, laying down the bases for the current applications in proteomics and glycomics. Advances in protein sample transformation prior to analysis have led to reduction of sample consumption and contamination, enhancing throughput. Within this context, and thanks to the availability of new high performing materials and technologies, increasingly more efficient and miniaturized enzyme-based analytical tools have been proposed to overcome shortcomings encountered in the in-solution enzymatic reactions (protein digestion and protein deglycosylation, for proteomics and glycomics, respectively). In this context, immobilized enzyme reactors (IMERs) and IMER-based platforms have been developed as promising approaches toward automation and higher analysis throughput. The scenario is in continuous development as underlined by thirty-four papers published in the last five years. This review encompasses recent advances in the design and operational set-ups of IMERs purposely developed for the analysis of proteins and glycoproteins. Recently developed dual IMERs, which integrate more than one processing step into a single IMER, and analytical platforms exploiting tandem IMERs are also reviewed and commented.
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http://dx.doi.org/10.1016/j.jpba.2018.07.051DOI Listing
October 2018

Exploiting the Chalcone Scaffold to Develop Multifunctional Agents for Alzheimer's Disease.

Molecules 2018 Jul 30;23(8). Epub 2018 Jul 30.

Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy.

Alzheimer's disease still represents an untreated multifaceted pathology, and drugs able to stop or reverse its progression are urgently needed. In this paper, a series of naturally inspired chalcone-based derivatives were designed as structural simplification of our previously reported benzofuran lead compound, aiming at targeting both acetyl (AChE)- and butyryl (BuChE) cholinesterases that, despite having been studied for years, still deserve considerable attention. In addition, the new compounds could also modulate different pathways involved in disease progression, due to the peculiar -α,β-unsaturated ketone in the chalcone framework. All molecules presented in this study were evaluated for cholinesterase inhibition on the human enzymes and for antioxidant and neuroprotective activities on a SH-SY5Y cell line. The results proved that almost all the new compounds were low micromolar inhibitors, showing different selectivity depending on the appended substituent; some of them were also effective antioxidant and neuroprotective agents. In particular, compound , endowed with dual AChE/BuChE inhibitory activity, was able to decrease ROS formation and increase GSH levels, resulting in enhanced antioxidant endogenous defense. Moreover, this compound also proved to counteract the neurotoxicity elicited by Aβ oligomers, showing a promising neuroprotective potential.
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http://dx.doi.org/10.3390/molecules23081902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222323PMC
July 2018

Isolation and Characterization of Wheat Derived Nonspecific Lipid Transfer Protein 2 (nsLTP2).

J Food Sci 2018 Jun 22;83(6):1516-1521. Epub 2018 May 22.

Dept. for Life Quality Studies, Alma Mater Studiorum Univ. of Bologna, Corso D'Augusto 237, 47921, Rimini, Italy.

Numerous studies support the protective role of bioactive peptides against cardiovascular diseases. Cereals represent the primary source of carbohydrates, but they also contain substantial amounts of proteins, therefore representing a potential dietary source of bioactive peptides with nutraceutical activities. The analysis of wheat extracts purified by chromatographic techniques by means of HPLC-UV/nanoLC-nanoESI-QTOF allowed the identification of a signal of about 7 kDa which, following data base searches, was ascribed to a nonspecific lipid-transfer protein (nsLTP) type 2 from Triticum aestivum (sequence coverage of 92%). For the first time nsLTP2 biological activities have been investigated. In particular, in experiments with human umbilical vein endothelial cells (HUVEC), nsLTP2 displayed antioxidant and cytoprotective activities, being able to significantly decrease reactive oxygen species (ROS) levels and to reduce lactate dehydrogenase (LDH) release, generated following oxidative (hydrogen peroxide) and inflammatory (tumor necrosis factor α, interleukin-1β, and lipopolysaccharide) stimulation. The obtained promising results suggest potential protective role of nsLTP2 in vascular diseases prevention. PRACTICAL APPLICATION: nsLTP 2 peptide is resistant to proteases throughout the gastrointestinal tract and exerts antioxidant and cytoprotective activities. These characteristics could be exploited in vascular diseases prevention.
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http://dx.doi.org/10.1111/1750-3841.14175DOI Listing
June 2018

A patent review of butyrylcholinesterase inhibitors and reactivators 2010-2017.

Expert Opin Ther Pat 2018 06;28(6):455-465

b Department of Pharmacy and Biotechnology , Alma Mater Studiorum Università di Bologna , Bologna , Italy.

Introduction: Butyrylcholinesterase (BuChE) has obtained a renewed interest as therapeutic target in Alzheimer's disease (AD), when changes in BuChE activity and expression along disease progression were highlighted as well as correlation between BuChE levels and cognitive function.

Areas Covered: During the last eight years, fourteen patents on BuChE inhibitors (BuChEI) have been submitted. Only three of them relate to BuChE selective inhibitors, while four of them focus on multitarget inhibitors which address different key pathological factors other than BuChE. Two patents report on non-selective acetylcholinesterase (AChE)/BuChE inhibitors, while four patents deal with natural compounds and their derivatives. One patent relates to antitoxic agents to treat exposure to ChEI pesticides and nerve agents.

Expert Opinion: Increasing evidence supports BuChE as a more beneficial target in moderate-to-severe forms of AD in comparison to the well-known AChE. However, hitting a single pathological target is likely not sufficient to halt the disease progression. Therefore, patented BuChE inhibitors with a multifunctional profile may open new therapeutic avenues, since the additional activities could reinforce the therapeutic effects. Unfortunately, in vivo studies are limited and key parameters, such as ADMET data, are missing. This lack of information makes difficult to forecast the development of patented BuChEIs into effective drug candidates.
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http://dx.doi.org/10.1080/13543776.2018.1476494DOI Listing
June 2018

Structural basis for the magnesium-dependent activation of transketolase from Chlamydomonas reinhardtii.

Biochim Biophys Acta Gen Subj 2017 Aug 24;1861(8):2132-2145. Epub 2017 May 24.

Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.

Background: In photosynthetic organisms, transketolase (TK) is involved in the Calvin-Benson cycle and participates to the regeneration of ribulose-5-phosphate. Previous studies demonstrated that TK catalysis is strictly dependent on thiamine pyrophosphate (TPP) and divalent ions such as Mg.

Methods: TK from the unicellular green alga Chlamydomonas reinhardtii (CrTK) was recombinantly produced and purified to homogeneity. Biochemical properties of the CrTK enzyme were delineated by activity assays and its structural features determined by CD analysis and X-ray crystallography.

Results: CrTK is homodimeric and its catalysis depends on the reconstitution of the holo-enzyme in the presence of both TPP and Mg. Activity measurements and CD analysis revealed that the formation of fully active holo-CrTK is Mg-dependent and proceeds with a slow kinetics. The 3D-structure of CrTK without cofactors (CrTK) shows that two portions of the active site are flexible and disordered while they adopt an ordered conformation in the holo-form. Oxidative treatments revealed that Mg participates in the redox control of CrTK by changing its propensity to be inactivated by oxidation. Indeed, the activity of holo-form is unaffected by oxidation whereas CrTK in the apo-form or reconstituted with the sole TPP show a strong sensitivity to oxidative inactivation.

Conclusion: These evidences indicate that Mg is fundamental to allow gradual conformational arrangements suited for optimal catalysis. Moreover, Mg is involved in the control of redox sensitivity of CrTK.

General Significance: The importance of Mg in the functionality and redox sensitivity of CrTK is correlated to light-dependent fluctuations of Mg in chloroplasts.
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http://dx.doi.org/10.1016/j.bbagen.2017.05.021DOI Listing
August 2017

Simple and rapid LC-MS method for the determination of circulating albumin microheterogeneity in veal calves exposed to heat stress.

J Pharm Biomed Anal 2017 Sep 25;144:263-268. Epub 2017 Apr 25.

Department of Veterinary Medical Sciences, University of Bologna, Bologna, Italy.

Heat stress has a major impact on veal calves welfare and productivity. Prolonged exposure to warm temperature is associated with several alterations of physiologic processes and increased systemic inflammation and oxidative stress. Bovine serum albumin (BSA) is the most abundant plasma protein and, besides the regulation of osmotic pressure, carries several additional functions, including antioxidant, immunomodulatory, binding and transport activities. Such non-oncotic properties are closely related to structural integrity of the circulating molecule and may be compromised in stressful microenvironments as it occurs in heat stressed animals. Thus, in the present study we developed and validated an LC-MS analytical technique for the characterization of circulating BSA microheterogeneity in veal calves exposed to heat stress. The method was specifically tailored to the structural characteristics of the BSA molecule as well as to the complexity of the biological samples, allowing the identification of several BSA isoforms, each characterized by a specific structural defect. The mass spectrometry based approach enabled the identification of BSA isoforms with reversible and irreversible oxidation and/or glycation and the native BSA, the only isoform endowed with structural and functional integrity. We found that, in veal calves, heat stress is associated to a significant reduction of the native BSA and to a significant increment of the reversibly and irreversibly oxidized BSA. Then, by monitoring the BSA microheterogeneity over a period of moderate heat stress, we found that the native BSA as well as the glycated BSA increased significantly during the recovery period. Based on our results the analysis of the BSA microheterogeneity could represent a novel biomarker for the assessment of animal welfare during environmental stressful conditions.
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http://dx.doi.org/10.1016/j.jpba.2017.04.041DOI Listing
September 2017

Quinazoline based α-adrenoreceptor antagonists with potent antiproliferative activity in human prostate cancer cell lines.

Eur J Med Chem 2017 Aug 4;136:259-269. Epub 2017 May 4.

Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy. Electronic address:

New α-adrenoreceptor (α-AR) antagonists related to prazosin and doxazosin were synthesized by replacing piperazine ring with (S)- or (R)-3-aminopiperidine. Binding studies indicated that the S configuration at the 3-C position of the piperidine ring is crucial for an optimal interaction of the compounds at all three α-AR subtypes. Quinazolines 9 and 10, bearing a quinone ring on the lateral chain, exhibited also potent antiproliferative activity in LNCaP androgen-sensitive prostate cancer cell lines, higher than that of doxazosin. Compound 10 increased apoptosis, in terms of DNA fragmentation, without triggering cell necrosis. The prooxidant activity found in compound 10 may underlie its ability to inhibit cell proliferation in synergy with the effect mediated by α-AR antagonism. Due to its better biological profile compared to doxazosin for LNCaP cell line, compound 10 might be a valuable lead compound for the design of new prostate antitumor agents.
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http://dx.doi.org/10.1016/j.ejmech.2017.05.003DOI Listing
August 2017

Structural and functional integrity of human serum albumin: Analytical approaches and clinical relevance in patients with liver cirrhosis.

J Pharm Biomed Anal 2017 Sep 18;144:138-153. Epub 2017 Apr 18.

Center for Applied Biomedical Research (C.R.B.A.), S. Orsola-Malpighi Hospital, Via Massarenti 9, 40138 Bologna, Italy; Department of Medical and Surgical Sciences, Alma Mater Studiorum University of Bologna, Via Massarenti 9, 40138, Italy. Electronic address:

Human serum albumin (HSA) is the most abundant circulating plasma protein. Besides a significant contribution to the osmotic pressure, it is also involved in the fine regulation of many other physiological processes, including the balance of the redox state, the inflammatory and/or immunological responses, and the pharmacokinetic and pharmacodynamics of many drugs. Growing evidence suggests that HSA undergoes structural and functional damage in diseases characterized by an enhanced systemic inflammatory response and oxidative stress, as it occurs in chronic liver disease. Based on their clinical relevance, this review provides a summary of the most common post-translational modifications affecting HSA structural integrity and functions and their clinical relevance in the field of liver disease. The review also provides a critical description of the analytical approaches employed for the investigation of conformational alterations and the identification/quantitation of specific post-translational modifications affecting HSA. Finally, the analytical methods available for the assessment of two of the most clinically relevant non-oncotic properties of HSA, namely the binding capacity and the antioxidant activity, are critically reviewed. Among the available techniques particular attention is given to those proposed for the in vitro and in vivo investigation of structurally modified albumin.
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http://dx.doi.org/10.1016/j.jpba.2017.04.023DOI Listing
September 2017

Towards automation in protein digestion: Development of a monolithic trypsin immobilized reactor for highly efficient on-line digestion and analysis.

Talanta 2017 May 5;167:143-157. Epub 2017 Feb 5.

Department of Pharmacy and Biotechnology, Alma Mater Studiorum - University of Bologna, via Belmeloro 6, 40126 Bologna, Italy.

Reducing experimental variability, limiting contamination and increasing automation are essential goals in the development of reliable analytical platforms for mass spectrometry (MS)-based proteomics. In this work novel trypsin-based monolithic immobilized enzyme reactors (tryp-IMERs), obtained by covalent immobilization on convective interaction media (CIMac™) analytical columns (5mm×5.2mm I.D.), were developed. Notwithstanding the small dimensions, column format allowed the insertion in common high performance liquid chromatography (HPLC) systems, thus avoiding the use of expensive micro- or nano-platforms. Monolith pore diameter and surface chemistry were optimized to achieve high digestion efficiency even with high molecular weight proteins and to avoid protein/peptide adsorption, peak broadening and sample loss. A full characterization of the tryp-IMERs was undertaken to select the best protocol for preparation and type of trypsin. Optimization of the operational and storage conditions was carried out by an off-line approach. On-line studies were performed by setting a multidimensional analytical platform, which included the tryp-IMER, a trapping column, an analytical C4 column and a high resolution hybrid mass spectrometer (ESI-Q-TOF). In the optimized conditions rapid protein digestion (90±9s), high protein coverage (≥60%) and high score values were achieved for five selected sample proteins (cytochrome c, myoglobin and albumins from different sources) differing in molecular size, isoelectric point and accessibility to cleavage sites as well as for a protein mixture of 200ng. The best performing tryp-IMERs showed high sensitivity down to the pmole level. The platform also resulted suitable for the analysis of high-molecular weight proteins such as a pool of human immunoglobulins G (hIgG) and for the high molecular weight fraction of human plasma proteins, which were digested in less than two minutes to an extent similar to that achieved by overnight incubation in a classical in solution protocol. Finally, underestimated key procedural issues were also highlighted during the study. Such aspects are of general interest both for tryp-IMER users and tryp-IMER developers.
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http://dx.doi.org/10.1016/j.talanta.2017.02.016DOI Listing
May 2017

Application of an ESI-QTOF method for the detailed characterization of GSK-3β inhibitors.

J Pharm Biomed Anal 2017 Sep 27;144:159-166. Epub 2017 Feb 27.

Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Rimini, Italy. Electronic address:

The crucial role of Glycogen Synthase Kinase 3 (GSK-3β) as a pivotal player in Alzheimer's Disease (AD) has recently inspired significant attempts to design and synthesize potent kinase inhibitors. In fact GSK-3β is considered the main kinase which catalyzes the microtubule-associated protein tau hyper-phosphorylation and the neurofibrillary tangles (NFT) in vitro and in vivo, The first classes of GSK-3β inhibitors were classified as ATP-competitive and, therefore, they lack of an efficient degree of selectivity over other kinases. In light of this consideration, many efforts are devoted to characterize new non ATP-competitive GSK-3β inhibitors, endowed with high selectivity. In parallel, there is an urgent need to develop new analytical methodologies for the hit selection (highthroughput screening) and ligand binding characterization in terms of potency, affinity and mechanism of action. The new methodology for GSK-3β enzymatic activity determination can be adopted as a realistic alternative to the currently used radioactive, luminescence and fluorescence detection methods, each showing limitations in terms of safety and interferences. Herein, we propose an alternative and selective electrospray ionization quadrupole time-of-flight (ESI-QTOF) method, based on the direct quantification of phosphorylated substrate muscle glycogen synthase GSM, a peptide resembling the high affinity sequence of natural substrate muscle glycogen synthase 1, for the detailed characterization of GSK-3β inhibitors. The method was validated in terms of accuracy and reproducibility of GSM signal intensity with a relative standard deviation RSD% value of 3.55%; Limit of Detection (LOD): 0.006μM; Lower Limit of Quantification (LLOQ): 0.02μM; linearity r 0.9951. The kinetic constants (K and v) of the GSK-3β catalyzed kinase reaction and the inhibitory potency of known ligands (IC), were determined. All the obtained results were in agreement with those reported in literature or obtained in house by the standard reference luminometric approach. The proposed method was applied to the elucidation of well known inhibitors mechanism of action by the construction of a Lineweaver-Burk plot and the K determination. Furthermore, the potency, affinity and mechanism of action of a new non ATP-competitive compound were established. We demonstrated the ESI-QTOF method to be more feasible than the classic kinase assays since it avoids drawbacks inherently connected with radioisotope labeling or the indirect detection of kinase activity, so far. It is also scalable to the screening of large library collections and suitable for pharmaceutical industries purposes.
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http://dx.doi.org/10.1016/j.jpba.2017.02.036DOI Listing
September 2017

Albumin Homodimers in Patients with Cirrhosis: Clinical and Prognostic Relevance of a Novel Identified Structural Alteration of the Molecule.

Sci Rep 2016 10 26;6:35987. Epub 2016 Oct 26.

Department of Medical and Surgical Sciences, Alma Mater Studiorum University of Bologna, Italy.

Decompensated cirrhosis is associated to extensive post-transcriptional changes of human albumin (HA). This study aims to characterize the occurrence of HA homodimerization in a large cohort of patients with decompensated cirrhosis and to evaluate its association with clinical features and prognosis. HA monomeric and dimeric isoforms were identified in peripheral blood by using a HPLC-ESI-MS technique in 123 cirrhotic patients hospitalized for acute decompensation and 50 age- and sex-comparable healthy controls. Clinical and biochemical parameters were recorded and patients followed up to one year. Among the monomeric isoforms identified, the N- and C-terminal truncated and the native HA underwent homodimerization. All three homodimers were significantly more abundant in patients with cirrhosis, acute-on-chronic liver failure and correlate with the prognostic scores. The homodimeric N-terminal truncated isoform was independently associated to disease complications and was able to stratify 1-year survival. As a result of all these changes, the monomeric native HA was significantly decreased in patients with cirrhosis, being also associated with a poorer prognosis. In conclusion homodimerization is a novel described structural alteration of the HA molecule in decompensated cirrhosis and contributes to the progressive reduction of the monomeric native HA, the only isoform provided of structural and functional integrity.
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http://dx.doi.org/10.1038/srep35987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5080612PMC
October 2016

Direct determination of GSK-3β activity and inhibition by UHPLC-UV-vis diode arrays detector (DAD).

J Pharm Biomed Anal 2016 May 23;124:104-111. Epub 2016 Feb 23.

Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Rimini, Italy. Electronic address:

Altered GSK-3β activity can contribute to a number of pathological processes including Alzheimer's disease (AD). Indeed, GSK-3β catalyzes the hyperphosphorylation of tau protein by transferring a phosphate moiety from ATP to the protein substrate serine residue causing the formation of the toxic insoluble neurofibrillary tangles; for this reason it represents a key target for the development of new therapeutic agents for AD treatment. Herein we describe a new selective UHPLC methodology developed for the direct characterization of GSK-3β kinase activity and for the determination of its inhibition, which could be crucial in AD drug discovery. The UHPLC-UV (DAD) based method was validated for the very fast determination of ATP as reactant and ADP as product, and applied for the analysis of the enzymatic reaction between a phosphate primed peptide substrate (GSM), resembling tau protein sequence, ATP and GSK-3β, with/without inhibitors. Analysis time was ten times improved, when compared with previously published chromatographic methods. The method was also validated by determining enzyme reaction kinetic constants (KM and vmax) for GSM and ATP and by analyzing well known GSK-3β inhibitors. Inhibition potency (IC50) values for SB-415286 (81 ± 6 nM) and for Tideglusib (251 ± 17 nM), found by the newly developed UHPLC method, were in good agreement with the luminescence method taken as independent reference method. Further on, the UHPLC method was applied to the elucidation of Tideglusib mechanism of action by determining its inhibition constants (Ki). In agreement with literature data, Tideglusib resulted a GSM competitive inhibitor, whereas SB-415286 was found inhibiting GSK-3β in an ATP competitive manner. This method was applied to the determination of the potency of a new lead compound and was found potentially scalable to inhibitor screening of large compounds collections.
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http://dx.doi.org/10.1016/j.jpba.2016.02.029DOI Listing
May 2016

Mass spectrometry characterization of circulating human serum albumin microheterogeneity in patients with alcoholic hepatitis.

J Pharm Biomed Anal 2016 Apr 25;122:141-7. Epub 2016 Jan 25.

Department of Pharmacy and Biotechnology, Via Sand Donato 15, 40127, University of Bologna, Italy. Electronic address:

Human serum albumin (HSA) is the most abundant plasma protein, endowed with several biological properties unrelated to its oncotic power, such as antioxidant and free-radicals scavenging activities, binding and transport of many endogenous and exogenous substances, and regulation of endothelial function and inflammatory response. These non-oncotic activities are closely connected to the peculiarly dynamic structure of the albumin molecule. HSA undergoes spontaneous structural modifications, mainly by reaction with oxidants and saccharides; however, patients with cirrhosis show extensive post-transcriptional changes at several molecular sites of HSA, the degree of which parallels the severity of the disease. The present work reports the development and application of an innovative LC-MS analytical method for a rapid and reproducible determination of the relative abundance of HSA isoforms in plasma samples from alcoholic hepatitis (AH) patients. A condition of severe oxidative stress, similar to that observed in AH patients, is associated with profound changes in circulating HSA microheterogeneity. More interestingly, the high resolution provided by the analytical platform allowed the monitoring of novel oxidative products of HSA never reported before.
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http://dx.doi.org/10.1016/j.jpba.2016.01.048DOI Listing
April 2016