Publications by authors named "Carlo Bertucci"

88 Publications

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 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

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

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

Absolute Configuration Assignment of Chiral Resorcin[4]arenes from ECD Spectra.

J Org Chem 2017 01 19;82(1):202-210. Epub 2016 Dec 19.

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

Racemates of five chiral resorcin[4]arenes, four tetra-O-substituted and one hepta-O-substituted, have been resolved by enantioselective HPLC, and their ECD spectra have been recorded online by stopped-flow measurements. The absolute configuration has been assigned by comparison of the experimental ECD spectra with DFT and semiempirical calculations. For the four tetra-O-substituted resorcin[4]arenes, the ECD exciton couplet at longer wavelength depends on the chirality induced in the arene scaffold by the substituents rather than on the precise nature of the substituents themselves. Accordingly, the exciton chirality model with excitons localized on the arene scaffold, here generalized to C symmetry, accurately describes the relationship between stereochemistry and chiroptical properties for this couplet, while its application at shorter wavelengths is unsafe. For the significantly larger hepta-O-substituted system the assignment particularly benefits from the use of the semiempirical ZINDO method.
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http://dx.doi.org/10.1021/acs.joc.6b02349DOI Listing
January 2017

Human Serum Albumin as Chiral Selector in Enantioselective High-Performance Liquid Chromatography.

Curr Med Chem 2017 ;24(8):743-757

Department of Pharmacy and Biotechnology, University of Bologna, via Belmeloro 6, I-40126 Bologna, Italy.

Enantioselective high-performance liquid chromatography (eHPLC) using chiral stationary phases (CSPs) is surely the most used technique for the determination of the enantiomeric excess (e.e.) of chiral drugs, a fundamental parameter for reliable studies on the relationship between stereochemistry and pharmacological activity. A key aspect of this enantioseparation technique is the efficiency of the chiral selector, which can be optimized to obtain higher selectivity and a wider applicability. Thus, the determination of the mechanisms behind chiral recognition is very important to predict and improve the enantioselectivity of CSPs. The present review deals with the preparation and use of CSPs for eHPLC with human serum albumin (HSA) as chiral selector, with particular emphasis on the modulation of the chromatographic performance. HSA-based CSPs allow a relatively easy prediction of the binding sites involved in the retention of analytes and the possibility to improve the selectivity of enantioresolution by modulating the binding process, using either reversible or covalent modifications of the protein. Significant improvements of the chromatographic parameters, such as reduction of analysis time and increase of enantioselectivity, have been obtained for selected analytes by using competitors for a particular binding site of HSA dissolved in the mobile phase or by selectively modifying the protein structure at single amino acid residues.
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http://dx.doi.org/10.2174/0929867324666161118115711DOI Listing
July 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

Surface plasmon resonance and isothermal titration calorimetry to monitor the Ni(II)-dependent binding of Helicobacter pylori NikR to DNA.

Anal Bioanal Chem 2016 Nov 31;408(28):7971-7980. Epub 2016 Aug 31.

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

NikR is a transcription factor that regulates the expression of Ni(II)-dependent enzymes and other proteins involved in nickel trafficking. In the human pathogenic bacterium Helicobacter pylori, NikR (HpNikR) controls, among others, the expression of the Ni(II) enzyme urease by binding the double-strand DNA (dsDNA) operator region of the urease promoter (OP ) in a Ni(II)-dependent mode. This article describes the complementary use of surface plasmon resonance (SPR) spectroscopy and isothermal titration calorimetry (ITC) to carry out a mechanistic characterization of the HpNikR-OP interaction. An active surface was prepared by affinity capture of OP and validated for the recognition process in the SPR experiments. Subsequently, the Ni(II)-dependent affinity of the transcription factor for its operator region was assessed through kinetic evaluation of the binding process at variable Ni(II) concentrations. The kinetic data are consistent with a two-step binding mode involving an initial encounter between the two interactants, followed by a conformational rearrangement of the HpNikR-OP complex, leading to high affinity binding. This conformational change is only observed in the presence of the full set of four Ni(II) ions bound to the protein. The SPR assay developed and validated in this study constitutes a suitable method to screen potential drug lead candidates acting as inhibitors of this protein-dsDNA interaction. Graphical Abstract Pictorial representation of the interaction between HpNikR, flowing in solution, and the OP urease promoter immobilized on the sensor chip surface.
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http://dx.doi.org/10.1007/s00216-016-9894-0DOI Listing
November 2016

Development and Pharmacological Characterization of Selective Blockers of 2-Arachidonoyl Glycerol Degradation with Efficacy in Rodent Models of Multiple Sclerosis and Pain.

J Med Chem 2016 03 7;59(6):2612-32. Epub 2016 Mar 7.

European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena , via Aldo Moro 2, I-53100 Siena, Italy.

We report the discovery of compound 4a, a potent β-lactam-based monoacylglycerol lipase (MGL) inhibitor characterized by an irreversible and stereoselective mechanism of action, high membrane permeability, high brain penetration evaluated using a human in vitro blood-brain barrier model, high selectivity in binding and affinity-based proteomic profiling assays, and low in vitro toxicity. Mode-of-action studies demonstrate that 4a, by blocking MGL, increases 2-arachidonoylglycerol and behaves as a cannabinoid (CB1/CB2) receptor indirect agonist. Administration of 4a in mice suffering from experimental autoimmune encephalitis ameliorates the severity of the clinical symptoms in a CB1/CB2-dependent manner. Moreover, 4a produced analgesic effects in a rodent model of acute inflammatory pain, which was antagonized by CB1 and CB2 receptor antagonists/inverse agonists. 4a also relieves the neuropathic hypersensitivity induced by oxaliplatin. Given these evidence, 4a, as MGL selective inhibitor, could represent a valuable lead for the future development of therapeutic options for multiple sclerosis and chronic pain.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01812DOI Listing
March 2016

Surface plasmon resonance and circular dichroism characterization of cucurbitacins binding to serum albumins for early pharmacokinetic profiling.

J Pharm Biomed Anal 2016 Apr 27;122:166-72. Epub 2016 Jan 27.

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

Cucurbitacins are a group of tetracyclic triterpenoids, known for centuries for their anti-cancer and anti-inflammatory properties, which are being actively investigated over the past decades in order to elucidate their mechanism of action. In perspective of being used as therapeutic molecules, a pharmacokinetic characterization is crucial to assess the affinity toward blood carrier proteins and extrapolate distribution volumes. Usually, pharmacokinetic data are first collected on animal models and later translated to humans; therefore, an early characterization of the interaction with carrier proteins from different species is highly desirable. In the present study, the interactions of cucurbitacins E and I with human and rat serum albumins (HSA and RSA) were investigated by means of surface plasmon resonance (SPR)-based optical biosensing and circular dichroism (CD) spectroscopy. Active HSA and RSA sensor chip surfaces were prepared through an amine coupling reaction protocol, and the equilibrium dissociation constants (Kd) for the different cucurbitacins-serum albumins complexes were then determined by SPR analysis. Further information on the binding of cucurbitacins to serum albumins was obtained by CD competition experiments with biliverdin, a specific marker binding to subdomain IB of HSA. SPR data unveiled a previously unreported binding event between CucI and HSA; the determined binding affinities of both compounds were slightly higher for RSA with respect to HSA, even though all the compounds can be ranked as high-affinity binders for both carriers. CD analysis showed that the two cucurbitacins modify the binding of biliverdin to serum albumins through opposite allosteric modulation (positive for HSA, negative for RSA), confirming the need for caution in the translation of pharmacokinetic data across species.
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http://dx.doi.org/10.1016/j.jpba.2016.01.051DOI Listing
April 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

Stopped-Flow Enantioselective HPLC-CD Analysis and TD-DFT Stereochemical Characterization of Methyl Trans-3-(3,4-Dimethoxyphenyl)Glycidate.

Chirality 2015 Dec 8;27(12):914-8. Epub 2015 Oct 8.

Department of Pharmacy and Biotechnology, Alma Mater Studiorum, University of Bologna, Bologna, Italy.

Caffeic acid-derived polyethers are a class of natural products isolated from the root extracts of comfrey and bugloss, which are endowed with intriguing pharmacological properties as anticancer agents. The synthesis of new polyether derivatives is achieved through ring-opening polymerization of chiral 2,3-disubstituted oxiranes, whose absolute configurations define the overall stereochemistry of the produced polymer. The absolute stereochemistry of one of these building blocks, methyl trans-3-(3,4-dimethoxy-phenyl)glycidate (3), was therefore characterized by the combination of enantioselective high-performance liquid chromatography (HPLC), electronic circular dichroism (ECD) spectroscopy, and time-dependent density functional theory (TD-DFT) calculations. Initial efforts aiming at the isolation of enantiomers by means of a standard preparative HPLC protocol followed by offline ECD analysis failed due to unexpected degradation of the samples after collection. The stopped-flow HPLC-CD approach, by which the ECD spectra of enantiomers are measured online with the HPLC system, was applied to overcome this issue and allowed a fast, reliable, and chemical-saving analysis, while avoiding the risks of sample degradation during the collection and processing of enantiomeric fractions. Subsequent TD-DFT calculations identified ( as the first eluted enantiomeric fraction on the Lux Cellulose-2 column, therefore achieving a full stereochemical characterization of the chiral oxirane under investigation.
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http://dx.doi.org/10.1002/chir.22539DOI Listing
December 2015

Ischaemia-modified albumin: a marker of bacterial infection in hospitalized patients with cirrhosis.

Liver Int 2015 Nov 21;35(11):2425-32. Epub 2015 May 21.

U.O. Semeiotica Medica, Department of Medical and Surgical Sciences, Alma Mater Studiorum University of Bologna, Bologna, Italy.

Background & Aims: Patients with cirrhosis present structural changes of human serum albumin (HSA) affecting non-oncotic functions. Ischaemia-modified albumin (IMA), which reflects the capacity to bind cobalt, has been associated to patient mortality during acute-on-chronic liver failure. This study aimed to assess whether circulating IMA is elevated in advanced cirrhosis and its relationship with severity of cirrhosis and specific complications.

Methods: A total of 127 cirrhotic patients hospitalized for an acute complication of the disease and 44 healthy controls were enrolled. Plasma IMA and IMA to albumin ratio (IMAr) were measured with a cobalt-binding assay. HSA isoforms carrying post-transcriptional molecular changes were assessed with HPLC-ESI-MS. The effect of endotoxemia on IMA was evaluated in rats with CCl4 -cirrhosis.

Results: IMA/IMAr is significantly higher in cirrhotic patients than in controls, but no correlations were found with prognostic scores. IMA did not correlate with the altered HSA isoforms. Ascites, renal impairment and hepatic encephalopathy did not influence IMA/IMAr levels. In contrast, IMA/IMAr is significantly higher in infected than non-infected patients. ROC curves showed that IMA/IMAr had similar discriminating performances for bacterial infection as C-reactive protein (CRP). Moreover, CRP and IMA were independently associated with bacterial infection. Consistently, endotoxin injection significantly increased IMA in cirrhotic, but not in healthy rats.

Conclusions: IMA is elevated in patients with advanced cirrhosis. The IMA level does not correlate with disease severity scores, but it is specifically associated to bacterial infection, showing a discriminating performance similar to CRP. Further investigations to assess IMA as a novel diagnostic test for bacterial infection are advocated.
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http://dx.doi.org/10.1111/liv.12860DOI Listing
November 2015

Species-dependent binding of new synthesized bicalutamide analogues to albumin by optical biosensor analysis.

J Pharm Biomed Anal 2015 17;111:324-32. Epub 2015 Feb 17.

Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy. Electronic address:

The binding of some novel bicalutamide analogues to human serum albumin (HSA) and rat serum albumin (RSA) was investigated by surface plasmon resonance (SPR) based optical biosensor technique. The serum protein binding of the bicalutamide analogues was determined and compared to that of the parent compound. Furthermore, HSA and RSA were used as target plasma proteins, in order to highlight possible differences among species when performing pharmacokinetic studies. HSA and RSA were covalently immobilized on carboxymethyl dextran matrixes, using an amine coupling procedure. The anchoring method was validated by determining the dissociation constant (KD) of a standard analyte to confirm that the binding properties of the proteins were maintained. The ranking of the bicalutamide analogues for their HSA and RSA bound fractions was used to compare the behaviour of the two albumins. Most of the bicalutamide analogues showed higher binding levels with respect to the lead compound, (R)-bicalutamide. Further, meaningful differences in the binding level to the two serum proteins were obtained. The dissociation constants (KD) of the interaction between the lead compound, (R)-bicalutamide, and the two proteins were calculated. As a result, the KD obtained with HSA was one order of magnitude higher than that obtained with RSA. The observed differences in the HSA and RSA bonding of the bicalutamide analogues increase the knowledge on the possible low reliability in extrapolating the distribution data obtained on animals to humans. This work demonstrates that SPR based optical biosensor technique is well suited for the medium-high throughput screening of compounds' ligand binding to serum albumins.
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http://dx.doi.org/10.1016/j.jpba.2015.02.010DOI Listing
February 2016

Induced circular dichroism as a tool to investigate the binding of drugs to carrier proteins: Classic approaches and new trends.

J Pharm Biomed Anal 2015 Sep 19;113:34-42. Epub 2015 Feb 19.

Department of Pharmacy and Biotechnology, University of Bologna, via Belmeloro 6, 40126 Bologna, Italy. Electronic address:

Induced circular dichroism (ICD) is a spectroscopic phenomenon that provides versatile and useful methods for characterizing the structural and dynamic properties of the binding of drugs to target proteins. The understanding of biorecognition processes at the molecular level is essential to discover and validate new pharmacological targets, and to design and develop new potent and selective drugs. The present article reviews the main applications of ICD to drug binding studies on serum carrier proteins, going from the classic approaches for the derivation of drug binding parameters and the identification of binding sites, to an overview of the emerging trends for the characterization of binding modes by means of quantum chemical (QC) techniques. The advantages and limits of the ICD methods for the determination of binding parameters are critically reviewed; the capability to investigate the binding interactions of drugs and metabolites to their target proteins is also underlined, as well as the possibility of characterizing the binding sites to obtain a complete picture of the binding mechanism and dynamics. The new applications of ICD methods to identify stereoselective binding modes of drug/protein complexes are then reviewed with relevant examples. The combined application of experimental ICD spectroscopy and QC calculations is shown to identify qualitatively the bound conformations of ligands to target proteins even in the absence of a detailed structure of the binding sites, either obtained from experimental X-ray crystallography and NMR measurements or from computational models of the complex.
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http://dx.doi.org/10.1016/j.jpba.2015.02.024DOI Listing
September 2015

A SMYD3 Small-Molecule Inhibitor Impairing Cancer Cell Growth.

J Cell Physiol 2015 Oct;230(10):2447-2460

Division of Medical Genetics, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari "Aldo Moro", Bari, Italy.

SMYD3 is a histone lysine methyltransferase that plays an important role in transcriptional activation as a member of an RNA polymerase complex, and its oncogenic role has been described in different cancer types. We studied the expression and activity of SMYD3 in a preclinical model of colorectal cancer (CRC) and found that it is strongly upregulated throughout tumorigenesis both at the mRNA and protein level. Our results also showed that RNAi-mediated SMYD3 ablation impairs CRC cell proliferation indicating that SMYD3 is required for proper cancer cell growth. These data, together with the importance of lysine methyltransferases as a target for drug discovery, prompted us to carry out a virtual screening to identify new SMYD3 inhibitors by testing several candidate small molecules. Here we report that one of these compounds (BCI-121) induces a significant reduction in SMYD3 activity both in vitro and in CRC cells, as suggested by the analysis of global H3K4me2/3 and H4K5me levels. Of note, the extent of cell growth inhibition by BCI-121 was similar to that observed upon SMYD3 genetic ablation. Most of the results described above were obtained in CRC; however, when we extended our observations to tumor cell lines of different origin, we found that SMYD3 inhibitors are also effective in other cancer types, such as lung, pancreatic, prostate, and ovarian. These results represent the proof of principle that SMYD3 is a druggable target and suggest that new compounds capable of inhibiting its activity may prove useful as novel therapeutic agents in cancer treatment.
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http://dx.doi.org/10.1002/jcp.24975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988495PMC
October 2015

Characterization of the species-dependent ketoprofen/albumin binding modes by induced CD spectroscopy and TD-DFT calculations.

J Pharm Biomed Anal 2015 Aug 22;112:176-80. Epub 2014 Nov 22.

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

The stereospecificity of high-affinity biorecognition phenomena at the basis of the activity of drugs is an important topic of active research in medicinal chemistry. The binding of drugs to their targets or to carrier proteins may lead to the onset of an induced circular dichroism (ICD) signal, which can be detected experimentally. Quantum mechanical (QM) calculations based on density functional theory (DFT) and its time-dependent formulation (TD-DFT) can be used to determine the theoretical chiroptical response of all the possible conformations of drugs bound to their hosts; by comparison with the experimental ICD spectra of drug-host complexes, this approach can lead to the identification of possible binding modes in the absence of X-ray crystallography or NMR data. The present article reports the application of experimental electronic circular dichroism (ECD) spectroscopy, DFT conformational analysis and TD-DFT calculations to the investigation of the binding modes of (S)-ketoprofen to serum albumins. The peculiar species-dependent ICD spectra observed for the binding of (S)-ketoprofen to different serum albumins can be explained by the selection of different mutual arrangements of the phenyl moieties inside the binding pocket. Such structural elucidations contribute to a better understanding of the changes in the pharmacokinetic and pharmacodynamic profiles of drugs among different species.
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http://dx.doi.org/10.1016/j.jpba.2014.11.029DOI Listing
August 2015

Mass spectrometric characterization of human serum albumin dimer: A new potential biomarker in chronic liver diseases.

J Pharm Biomed Anal 2015 Aug 10;112:169-75. Epub 2014 Dec 10.

Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna, Italy. Electronic address:

Human serum albumin (HSA) undergoes several structural alterations affecting its properties in pro-oxidant and pro-inflammatory environments, as it occurs during liver cirrhosis. These modifications include the formation of albumin dimers. Although HSA dimers were reported to be an oxidative stress biomarker, to date nothing is known about their role in liver cirrhosis and related complications. Additionally, no high sensitive analytical method was available for HSA dimers assessment in clinical settings. Thus the HSA dimeric form in human plasma was characterized by mass spectrometry using liquid chromatography tandem mass spectrometry (LC-ESI-Q-TOF) and matrix assisted laser desorption time of flight (MALDI-TOF) techniques. N-terminal and C-terminal truncated HSA, as well as the native HSA, undergo dimerization by binding another HSA molecule. This study demonstrated the presence of both homo- and hetero-dimeric forms of HSA. The dimerization site was proved to be at Cys-34, forming a disulphide bridge between two albumin molecules, as determined by LC-MS analysis after tryptic digestion. Interestingly, when plasma samples from cirrhotic subjects were analysed, the dimer/monomer ratio resulted significantly increased when compared to that of healthy subjects. These isoforms could represent promising biomarkers for liver disease. Additionally, this analytical approach leads to the relative quantification of the residual native HSA, with fully preserved structural integrity.
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http://dx.doi.org/10.1016/j.jpba.2014.12.001DOI Listing
August 2015

Short-range solvation effects on chiroptical properties: a time-dependent density functional theory and ab initio molecular dynamics computational case study on austdiol.

J Phys Chem A 2014 Dec 5;118(50):11751-7. Epub 2014 Dec 5.

Department of Pharmacy and Biotechnology, University of Bologna , Via Belmeloro 6, I-40126 Bologna, Italy.

The description of solvation effects on the chiroptical properties of chiral molecules is still a difficult challenge in the field of computational spectroscopy; this issue is critical in stereochemical characterization, since a reliable assessment of absolute configuration requires high accuracy. The present case study reports the huge effect of solvation on the chiroptical properties of austdiol, a fungal metabolite of known stereochemistry. Standard protocols based on time-dependent density functional theory calculations failed to reproduce its experimental chiroptical properties in methanol. When short-range solvation effects are explicitly considered by means of ab initio molecular dynamics, the correlation between calculated and experimental data is greatly improved because of a better description of the chiral environment around the ketone chromophore, showing that the modeling of subtle solvent-induced perturbations may require the most accurate computational methods.
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http://dx.doi.org/10.1021/jp511428vDOI Listing
December 2014

Determination of levamisole and tetramisole in seized cocaine samples by enantioselective high-performance liquid chromatography and circular dichroism detection.

J Chromatogr A 2014 Oct 29;1363:150-4. Epub 2014 Jul 29.

Department for Life Quality Studies, University of Bologna, Corso d'Augusto 237, 47921 Rimini, Italy.

Levamisole, an anthelmintic drug, has been increasingly employed as an adulterant of illicit street cocaine over the last decade; recently, the use of tetramisole, the racemic mixture of levamisole and its enantiomer dexamisole, was also occasionally observed. A new enantioselective high-performance liquid chromatography (HPLC) method, performed on cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phases in normal-phase mode, was validated to determine the enantiomeric composition of tetramisole enantiomers in seized cocaine samples. Furthermore, the hyphenation of the validated HPLC method with a circular dichroism (CD) detection system allowed the direct determination of elution order and a selective monitoring of levamisole and dexamisole in the presence of possible interferences. The method was applied to the identification and quantitation of the two enantiomers of tetramisole in seized street cocaine samples.
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http://dx.doi.org/10.1016/j.chroma.2014.07.069DOI Listing
October 2014

Posttranscriptional changes of serum albumin: clinical and prognostic significance in hospitalized patients with cirrhosis.

Hepatology 2014 Dec 3;60(6):1851-60. Epub 2014 Nov 3.

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

Unlabelled: Beside the regulation of fluid distribution, human serum albumin (HSA) carries other activities, such as binding, transport, and detoxification of many molecules. In patients with cirrhosis, HSA exhibits posttranscriptional alterations that likely affect its functions. This study aimed at identifying the structural HSA alterations occurring in cirrhosis and determining their relationship with specific clinical complications and patient survival. One hundred sixty-eight patients with cirrhosis, 35 with stable conditions and 133 hospitalized for acute clinical complications, and 94 healthy controls were enrolled. Posttranscriptional HSA molecular changes were identified and quantified by using a high-performance liquid chromatography/electrospray ionization mass spectrometry technique. Clinical and biochemical parameters were also recorded and hospitalized patients were followed for up to 1 year. Seven HSA isoforms carrying one or more posttranscriptional changes were identified. Altered HSA isoforms were significantly more represented in patients than in healthy controls. Conversely, the native, unchanged HSA isoform was significantly reduced in cirrhosis. Native HSA and most altered isoforms correlated with both Child-Pugh and Model for End-Stage Liver Disease scores. In hospitalized patients, oxidized and N-terminal truncated isoforms were independently associated with ascites, renal impairment, and bacterial infection. Finally, the native HSA and cysteinylated/N-terminal truncated isoforms were predictors of 1-year survival, with greater prognostic accuracy than total serum albumin concentration.

Conclusions: Extensive posttranscriptional changes of HSA, involving several molecular sites and increasing in parallel with disease severity, occur in patients with cirrhosis. Altered isoforms are independently associated with specific clinical complications, whereas the residual, native HSA isoform independently predicts patient survival. These findings support the concept of the "effective albumin concentration," which implies that the global HSA function is related not only to its serum concentration, but also to the preservation of its structural integrity.
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http://dx.doi.org/10.1002/hep.27322DOI Listing
December 2014

Enantioresolution, stereochemical characterization and biological activity of a chiral large-conductance calcium-activated potassium channel opener.

J Chromatogr A 2014 Oct 12;1363:162-8. Epub 2014 Jun 12.

Department of Pharmaceutical Sciences, University of Perugia, via Fabretti 48, 06123 Perugia, Italy. Electronic address:

A number of large-conductance calcium-activated potassium (BK) channel openers based on the 2-aryl-1,4-benzothiazine scaffold were previously synthesized, and 2-(5-bromo-2-methoxyphenyl)-6-trifluoromethyl-2H-1,4-benzothiazin-3(4H)-one (1) was identified as the most active compound. Since a stereoselective activation of BK channels was demonstrated for arylindolone derivatives, the effect of the absolute configuration at the C-2 position on the vasorelaxing potency of 2-aryl-1,4-benzothiazines is investigated in this article. Compound 1 was initially evaluated as a racemate: subsequently, the "racemic approach" was used to isolate its enantiomers. The excellent enantioresolution obtained using the Sepapak-4 column (CSP 4, cellulose tris(4-chloro-3-metylphenylcarbamate); RS=8.36; α=2.03) allowed to collect highly pure enantiomeric fractions, with enantiomeric excess (e.e.) values higher than 97% and 98% for the first- and second-eluted enantiomer, respectively. Electronic circular dichroism (ECD) studies on the two isolated enantiomers, combined with time-dependent density functional theory (TD-DFT) calculations allowed to characterize the configuration of the enantiomers and determine a (R), (S) elution order. Results from biological assays indicated that the racemate and the isolated enantiomers are endowed with comparable vasorelaxing potency.
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http://dx.doi.org/10.1016/j.chroma.2014.06.020DOI Listing
October 2014

Optical biosensor analysis in studying new synthesized bicalutamide analogs binding to androgen receptor.

J Pharm Biomed Anal 2014 Jul 12;95:151-7. Epub 2014 Mar 12.

Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy. Electronic address:

Bicalutamide (Casodex®) is a non-steroidal anti-androgen drug used in the treatment of prostate cancer, which represents the second most common malignancy diagnosed in men worldwide. In this work, we analyze the ability of some novel bicalutamide analogs to bind the androgen receptor, by using an optical biosensor. Androgen receptor was covalently immobilized on a carboxy methyl dextran matrix. The immobilized receptor chip was then used for the binding experiments of the bicalutamide analogs. The (R)-bicalutamide dissociation constant was in good agreement to the value reported in literature obtained by using radiolabeled targets. Most of the new synthesized compounds showed higher androgen receptor binding level, when compared to the reference. Our results clearly indicate that the surface plasmon resonance (SPR) technique offers many advantages with respect to other available technologies in terms of studying biomolecular interactions. Moreover, this study provides an effective methodology for determining the binding affinity of novel chemical entities for the isolated androgen receptor, thus excluding possible off-target interactions occurring in conventional cell-based techniques.
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http://dx.doi.org/10.1016/j.jpba.2014.03.002DOI Listing
July 2014

N-aryl-2,6-dimethylbenzamides, a new generation of tocainide analogues as blockers of skeletal muscle voltage-gated sodium channels.

J Med Chem 2014 Mar 7;57(6):2589-600. Epub 2014 Mar 7.

Dipartimento di Farmacia-Scienze del Farmaco, ‡Sezione di Farmacologia, Università degli Studi di Bari "Aldo Moro" , via E. Orabona n. 4, 70126 Bari, Italy.

On the basis of a 3D-QSAR study, a new generation of tocainide analogues were designed and synthesized as voltage-gated skeletal muscle sodium channel blockers. Data obtained by screening new compounds by means of Hille-Campbell Vaseline gap voltage-clamp recordings showed that the elongation of the alkyl chain and the introduction of lipophilic and sterically hindered groups on the amino function enhance both potency and use-dependent block. The results provide additional indications about the structural requirement of pharmacophores for further increasing potency and state-dependent block and allowed us to identify a new tocainide analogue (6f) with a favorable pharmacodynamic profile to be proposed as a valid candidate for studies aimed at evaluating its usefulness in the treatment of myotonias.
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http://dx.doi.org/10.1021/jm401864bDOI Listing
March 2014

Species-dependent binding of tocainide analogues to albumin: affinity chromatography and circular dichroism study.

J Chromatogr B Analyt Technol Biomed Life Sci 2014 Oct 10;968:69-78. Epub 2014 Jan 10.

Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy. Electronic address:

A series of novel tocainide analogues were characterized for their HSA and RSA binding, by using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). In this HPLAC study, HSA and RSA were covalently immobilized to the silica matrix of HPLC columns, with a procedure that maintained unaltered the binding properties of the proteins. The tocainide analogues were ranked for their affinity to HSA and RSA on the basis of their bound fractions measured by the two albumin-based columns. This technique was also applied to characterize the high affinity binding sites of these tocainide analogues to the protein. For this purpose displacement experiments were carried out by means of increasing concentrations in the mobile phase of competitors known to bind selectively to the main binding sites of HSA. The results obtained with the immobilized proteins were confirmed by investigating the same drug-protein systems in solution by circular dichroism. The comparison of the data collected with both methodologies highlighted the dramatic effect of small differences in the amino acidic sequences of the two proteins. In fact, despite their similar primary and secondary structures, a small difference in the amino acidic sequence leads to significant differences in their three-dimensional structure reflecting their different binding capacity and their stereoselectivity. Therefore, this study confirms how it is crucial to consider the significant differences among the animal models when performing pharmacokinetic studies. It is also clear that the knowledge of serum carrier binding parameters at an early stage of drug discovery represents a great advantage that may help to save time and efforts.
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http://dx.doi.org/10.1016/j.jchromb.2014.01.007DOI Listing
October 2014

Stereochemical and conformational study on fenoterol by ECD spectroscopy and TD-DFT calculations.

J Pharm Biomed Anal 2014 Mar 30;91:92-6. Epub 2013 Dec 30.

Department of Pharmacy and Biotechnology, University of Bologna, via Belmeloro 6, 40126 Bologna, Italy. Electronic address:

Fenoterol and its derivatives are selective β2-adrenergic receptor (β2-AR) agonists whose stereoselective biological activities have been extensively investigated in the past decade; a complete stereochemical characterization of fenoterol derivatives is therefore crucial for a better understanding of the effects of stereochemistry on β2-AR binding. In the present project, the relationship between chiroptical properties and absolute stereochemistry of the stereoisomers of fenoterol (1) was investigated by experimental ECD spectroscopy and time-dependent density functional theory (TD-DFT). DFT geometry optimizations were carried out at the RI-B97D/TZVP/IEFPCM(MeOH) level and subsequent TD-DFT calculations were performed using the PBE0 hybrid functional. Despite the large pool of equilibrium conformers found for the investigated compounds and the known limitations of the level of theory employed, the computational protocol was able to reproduce the experimental ECD spectra of the stereoisomers of 1. The main contribution to the overall chiroptical properties was found to arise from the absolute configuration of the chiral center in α-position to the resorcinol moiety. Based on this evidence, a thorough conformational analysis was performed on the optimized DFT conformers, which revealed the occurrence of a different equilibrium between conformational patterns for the diastereomers of fenoterol: the (R,R')/(S,S') enantiomeric pair showed a higher population of folded conformations than the (R,S')/(S,R') pair.
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http://dx.doi.org/10.1016/j.jpba.2013.12.018DOI Listing
March 2014

Mycoleptones A-C and polyketides from the endophyte Mycoleptodiscus indicus.

J Nat Prod 2014 Jan 3;77(1):70-8. Epub 2014 Jan 3.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo , 14040-903, Ribeirão Preto, SP, Brazil.

Three new azaphilones with an unusual methylene bridge, named mycoleptones A, B, and C (2, 4, and 5), were isolated from cultures of Mycoleptodiscus indicus, a fungus associated with the South American medicinal plant Borreria verticillata. Additionally, four known polyketides, austdiol (1), eugenitin (3), 6-methoxyeugenin (6), and 9-hydroxyeugenin (7), were also isolated. The structural characterization of compounds was carried out by nuclear magnetic resonance spectroscopy, high-resolution mass spectrometry, electronic circular dichroism spectroscopy, time-dependent density functional theory calculations, and X-ray crystallography. Compounds 1-9 were weakly active when tested in antileishmanial and cytotoxicity assays.
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http://dx.doi.org/10.1021/np4006822DOI Listing
January 2014

Determination of dextromethorphan and levomethorphan in seized heroin samples by enantioselective HPLC and electronic CD.

J Pharm Biomed Anal 2013 Jul-Aug;81-82:76-9. Epub 2013 Apr 6.

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

A new enantioselective HPLC method was developed for the resolution and determination of the enantiomers of methorphan, dextromethorphan (DXM) and levomethorphan (LVM), on a Chiralcel OJ column (250 mm × 4.6mm I.D.). The resolution of DXM and LVM was obtained using a mobile phase consisting of (n-hexane)-(2-propanol)-diethylamine (70:30:0.1, v/v/v) at a flow rate of 0.5 mL min(-1). The enantioselective method was found to be selective (α=1.92) and sensitive (LOD=2.8 μg mL(-1) for both DXM and LVM). The method was coupled with electronic circular dichroism (CD), allowing the determination of the elution order on the basis of the sign of CD signals of the single enantiomers at 285 nm (positive for DXM, negative for LVM). Under the optimized conditions, the validated method was applied to the identification and quantitation of the enantiomers of methorphan in samples of different sources of illicit drugs of abuse (heroin). DXM was found in eight seized samples of street heroin; two of these samples, for which the DXM content was found to be over 5% (w/w) and exceeding a 10% (w/w) ratio with respect to diacetylmorphine, were the cause of two deaths for overdose due to acute narcotism.
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http://dx.doi.org/10.1016/j.jpba.2013.03.024DOI Listing
December 2013